Epitope focusing by variable effective antigen surface concentration

ABSTRACT

The present disclosure provides compositions and methods for the generation of an antibody or immunogenic composition, such as a vaccine, through epitope focusing by variable effective antigen surface concentration. Generally, the composition and methods of the disclosure comprise three steps: a “design process” comprising one or more in silico bioinformatics steps to select and generate a library of potential antigens for use in the immunogenic composition; a “formulation process”, comprising in vitro testing of potential antigens, using various biochemical assays, and further combining two or more antigens to generate one or more immunogenic compositions; and an “administering” step, whereby the immunogenic composition is administered to a host animal, immune cell, subject or patient. Further steps may also be included, such as the isolation and production of antibodies raised by host immune response to the immunogenic composition.

This application is a continuation of U.S. application Ser. No.14/398,084, filed on Oct. 30, 2014, which is a U.S. National Stage Entryof International Application No. PCT/US2013/042098, filed May 21, 2013,which claims the benefit to U.S. Provisional Application 61/649,392,filed May 21, 2012 and U.S. Provisional Application 61/801,135, filedMar. 15, 2013, each of which are incorporated by reference in theirentirety.

BACKGROUND

Pathogenic agents such as, infectious bacteria, parasites, fungi,viruses and cancers, have evolved various strategies to evade detectionand neutralization by host immune response. Such strategies can oftenundermine and complicate the development of successful vaccines towardsthese pathogens. For example, certain parasites have evolved the abilityto enter intracellular habitats to avoid the effects of neutralizingantibodies circulating in the blood. Other pathogens, such astrypanosomes have evolved a process known as antigenic variation tochange the character of their surface coats. Similarly, pathogens suchas some bacteria and viruses have evolved mechanisms to introducegenetic variation in coding regions of their genomes, thereby generatingslight alterations to structures of proteins within the pathogen toevade binding by immune cell receptors. Slight changes in variableloops, changes in glycosylation patterns, oligomerization andconformational masking may help the pathogen evade detection andneutralization by immune response molecules such as antibodies.

In some cases, pathogens may display one or more immunodominantepitopes, prone to elicit immune response, but that undergo structuralvariation or antigenic drift. The host immune response raises earlyneutralizing antibodies against one or more of these immunodominantepitopes in an attempt to reduce the titer of the dominant pathogenicphenotype. The immunodominant epitopes may also serve to mask immuneresponse to other more conserved epitopes in the pathogen. Antigenicdrift of immunodominant epitopes results in these early neutralizingantibodies becoming ineffective against the pathogen. This may occurduring the course of a single infection, or over the course of multipleinfections.

With respect to human pathogens, the human immunodeficiency virus-1(HIV-1) provides an example of a highly effective strategy used toevade, and so to destroy, the human immune system. None of the vaccineapproaches that have been attempted to date have proved successful dueto the high prevalence of antigenic drift in multiple immunodominantepitopes of HIV-1 capsid proteins. Typical approaches using subunitvaccine strategies have failed, as the virus is able to evolve veryquickly to evade neutralization by antibodies raised during vaccination.

The influenza virus hemagglutinin antigen (HA) provides another exampleof a pathogen-encoded immunodominant antigen that is subject toantigenic drift. Variation in the antigenic structure of HA correlateswith the periodic epidemics of respiratory disease that are caused bythis virus, despite the widespread use of influenza vaccine.

There is need in the art for novel approaches for the generation ofimproved vaccines. Specifically, there is a need for methods to generateantibodies to specific or desired epitopes of a particular antigen. Insome cases, desired epitopes for vaccine development may be more highlyconserved than other immunodominant epitopes. Such methods may beapplied to the generation of antibodies for a wide range of antigens andprotein targets, or vaccines for numerous human diseases, such asinfections caused by viruses and microorganisms as well as cancer.

SUMMARY OF THE DISCLOSURE

In some aspects, the disclosure provides for an immunogenic compositionfor eliciting an immune response in a subject, the immunogeniccomposition comprising at least 6 antigens, wherein each antigencomprises a common target epitope, and wherein each antigen of theimmunogenic composition comprises an individual concentrationinsufficient to be immunogenic in the subject.

In some aspects, the disclosure provides for an immunogenic compositionfor eliciting an immune response in a subject, the immunogeniccomposition comprising at least 6 antigens, wherein each antigencomprises a common target epitope, and wherein each antigen of theimmunogenic composition comprises at least 100 amino acid residues.

In some aspects, the disclosure provides for an immunogenic compositionfor eliciting an immune response in a subject, the immunogeniccomposition comprising at least 6 antigens, wherein each antigencomprises: a common target epitope at least 90% identical across allantigens of the immunogenic composition; at most 90% identical sequencein surface exposed regions between any two antigen variants outside ofthe common target epitope.

In some aspects, the disclosure provides for a unit dose of animmunogenic composition comprising at least 6, antigens, wherein eachantigen comprises a common target epitope, and wherein each antigen ofthe immunogenic composition comprises an individual concentrationinsufficient to be immunogenic in the subject.

In some aspects, the disclosure provides for a method of generating animmune response to a target epitope, the method comprising: creatingwith a computing device, a computation-guided library comprising aplurality of antigen variants, wherein each antigen variant comprises: aconserved target epitope region; and one or more non conserved regions,wherein the non conserved regions are outside the target epitope;obtaining a plurality of antigen variants of the computation-guidedlibrary; generating an immunogenic composition comprising the pluralityof antigen variants, wherein an individual antigen variant is present inthe immunogenic composition at an concentration insufficient to beimmunogenic.

In some aspects, the immunogenic composition is contacted with an immunecell.

In some aspects, an antibody that binds the target epitope is isolatedfrom the immune cell.

In some aspects, the immune cell is administered for treating orreducing the likelihood of a disease in a human subject in need thereof.

In some aspects, the disclosure provides for a computer executablealgorithm for the generation of an immunogenic composition, thealgorithm comprising: obtaining a target antigen protein sequence andone or more antigen homolog sequences; obtaining structural models fromthe target antigen protein sequence and the one or more antigen homologsequences; aligning the structural models of target antigen and the oneor more antigen homologs; extracting a positional weight matrix (PWM) ofeach amino acid residue frequency of the antigen protein sequence usingthe alignment of (c); identifying surface exposed amino acid residues inthe PWM generated in (d); identifying amino acid residues of one or moretarget epitopes in the PWM generated in (d); identifying one or moretarget epitopes in the PWM generated in (d); generating a library of aplurality of antigen variants comprising: diversifying amino acidresidues in non target epitope surface exposed amino acid residues ofthe PWM for each antigen variant; performing pairwise comparisons of oneor more non target epitope surface exposed regions comprising at acommonly defined area between antigen variants of the library, whereinantigen variants of the library share at least 30% sequence identity innon surface exposed amino acid residues, and wherein antigen variants ofthe library share at most 90% sequence identity in non target epitopesurface exposed amino acid residues; selecting two or more antigenvariants generated in (h); generating an immunogenic compositioncomprising a plurality of antigen variants, wherein an individualantigen variant in the immunogenic composition is assigned aconcentration insufficient to be immunogenic.

In some aspects, the disclosure provides for a computing devicecomprising a processor; and data storage, storing instructions that,upon execution by the processor, cause the computing device to performfunctions comprising: obtaining a target antigen protein sequence andone or more antigen homolog sequences; obtaining structural models fromthe target antigen protein sequence and the one or more antigen homologsequences; aligning the structural models of target antigen and the oneor more antigen homologs; extracting a positional weight matrix (PWM) ofeach amino acid residue frequency of the antigen protein sequence usingthe alignment of (c); identifying surface exposed amino acid residues inthe PWM generated in (d); identifying amino acid residues of one or moretarget epitopes in the PWM generated in (d); identifying one or moretarget epitopes in the PWM generated in (d); generating a library of aplurality of antigen variants comprising: diversifying amino acidresidues in non target epitope surface exposed amino acid residues ofthe PWM for each antigen variant: performing pairwise comparisons of oneor more non target epitope surface exposed regions comprising a commonlydefined area between antigen variants of the library, wherein antigenvariants of the library share at least 30% sequence identity in nonsurface exposed amino acid residues, and wherein antigen variants of thelibrary share at most 90% sequence identity in non target epitopesurface exposed amino acid residues; selecting two or more antigenvariants generated in (h); generating an immunogenic compositioncomprising a plurality of antigen variants, wherein an individualantigen variant in the immunogenic composition is assigned aconcentration insufficient to be immunogenic.

In some aspects the commonly defined area is at least 25 Å² or is ovalin shape.

In some aspects the library of a plurality of antigen variants comprisesat least 1×10⁶ antigen variants.

In some aspects, the disclosure provides for a method for generating animmunogenic composition, the method comprising: introducing into a cell,a nucleic acid encoding a plurality of antigen proteins, wherein eachantigen comprises a common target epitope; isolating the plurality ofantigen proteins; generating an immunogenic composition comprising theplurality of antigen variants, wherein an individual antigen variant inthe immunogenic composition is assigned a concentration insufficient tobe immunogenic.

In some aspects, the disclosure provides for a kit comprisingintroducing into a cell, a nucleic acid encoding a plurality of antigenproteins, wherein each antigen comprises a common target epitope;isolating the plurality of antigen proteins; generating an immunogeniccomposition comprising the plurality of antigen variants, wherein anindividual antigen variant in the immunogenic composition is assigned aconcentration insufficient to be immunogenic.

In some aspects, the disclosure provides for a method for detecting thepresence or absence of an antibody, the method comprising: contactingthe immunogenic composition or one or more antigen variants of any ofthe preceding immunogenic compositions described herein, comprising oneor more epitopes, with a composition comprising an antibody, underconditions suitable for binding of the antibody to the one or moreepitopes; and detecting one or more epitopes complexed with theantibody.

In some aspects, the disclosure provides for any of the precedingimmunogenic compositions described herein for use in a diagnostic forexposure to a pathogen or immune threat.

In some aspects, the disclosure provides for a virus like particle (VLP)comprising an immunogenic composition of preceding immunogeniccompositions described herein.

In some aspects, the immune response elicited by the immunogeniccomposition is determined by an immunoassay.

In some aspects, the immune response elicited by the immunogeniccomposition is determined by nucleic acid sequencing.

In some aspects, the immunogenic composition elicits an antibody in aB-cell of an immunized subject, and wherein the antibody titers of animmunized subject compared to antibody titers of a non-immunized subjectare measured.

In some aspects, the immunogenic composition elicits the production ofantibodies.

In some aspects, the immunogenic composition elicits an adaptive immuneresponse, humoral immune response or an innate immune response.

In some aspects, the immune response is a CD4+ T-cell response,including a Th1, Th2 and Th17 response.

In some aspects, the immunogenic composition is administered fortreating or reducing the likelihood of a disease in human subject inneed thereof.

In some aspects, the disease is selected from the group consisting of:infectious disease, autoimmune disease, inflammatory disease,neurological disease, addiction, cardiovascular disease, endocrinedisease and cancer.

In some aspects, the antigen is selected from the group consisting of:pneumococcal antigens, tuberculosis antigens, anthrax antigens, HIVantigens, seasonal or epidemic flu antigens, influenzae antigens,Pertussis antigens, Staphylococcus aureus antigens, Meningococcalantigens, Haemophilus antigens, HPV antigens, or combinations thereof.

In some aspects, the antigen is selected from the group consisting of:protein, peptide, lipoprotein, lipid, carbohydrate, glycoprotein andantigen encoding nucleic acid.

In some aspects, the disclosure provides for recombinant expressionvectors comprising nucleic acids encoding a plurality of antigenvariants of the immunogenic composition of any of the precedingimmunogenic compositions described herein.

In some aspects, the subject is a human.

In some aspects, the antigens share a common protein fold.

In some aspects, non exposed surface residues are not diversified.

In some aspects, immunogenic composition comprises at least 2, 3, 5, 10,25, 50, 100, 200, 250, 500, 1000, 1500, 5000, or 10000 antigen variants.

In some aspects, the immunogenic composition elicits an antibody in aB-cell of an immunized subject, and wherein the antibody titers aremeasured to be at least 10-fold, 20-fold, 50-fold, 100-fold, or1000-fold greater in an immunized subject than in an non-immunizedsubject.

In some aspects, the immunogenic composition elicits an antibody in aB-cell of an immunized subject, and wherein the antibody affinity forthe immunogenic composition or one or more antigen variants of theimmunogenic composition is measured.

In some aspects, the antibody affinity is determined by measuring theequilibrium dissociation constant of the antibody for the immunogeniccomposition or the one or more antigen variants of the immunogeniccomposition.

In some aspects, the the immunogenic composition elicits an antibody inB cells, wherein the antibody is measured to have a binding equilibriumdissociation constant to the immunogenic composition, or the one or moreantigen variants of the immunogenic composition that is less than 10⁻⁷M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M.

In some aspects the immunogenic composition elicits a class-switchrecombination in B cells.

In some aspects the immunogenic composition elicits the antibody isotypeproduced by the B cells to switch from IgM to IgG.

In some aspects the immunogenic composition elicits an antibody in aB-cell of an immunized subject, and wherein the affinity maturation ofantigen-specific antibodies is measured.

In some aspects the immunogenic composition elicits the formation ofmemory B cells or long-lived plasma cells capable of producing largeamounts of high-affinity antibodies for extended periods of time.

In some aspects the immunogenic composition elicits a vigorous germinalcenter reaction in B cells.

In some aspects the immunogenic composition elicits one or more antibodyisotypes, and wherein one or more antibody isotypes are identified.

In some aspects the immunogenic composition elicits the production ofIgG isotype antibodies.

In some aspects the immunogenic composition elicits B cells isdetermined by analyzing antibody function in neutralization assays.

In some aspects the immunogenic composition elicits helper T cells asdetermined by antigen-induced production of cytokines by T cells.

In some aspects the stimulation of helper T cell is determined bymeasuring antigen-induced production of cytokines by T cells.

In some aspects the cytokine is IFNγ, IL-4, IL-2, or TNFα.

In some aspects the immunogenic composition elicits an immune responsefrom T cells, and wherein antigen-induced production of cytokines by Tcells is measured by ELISPOT assay.

In some aspects the immunogenic composition elicits T cells, whereinimmunized subjects comprise about 10-fold, 100-fold, 1000-fold,1000-fold more cytokine-producing cells than do naive controls.

In some aspects the immunogenic composition elicits T cells, whereinantigen-induced production of cytokines by T cells is measured bydetermining antigen-induced proliferation of T cells.

In some aspects the immunogenic composition elicits T cells, wherein thestimulation of an immune response in T cells is determined by measuringcellular markers of T cell activation.

In some aspects the immunogenic composition elicits B cells, wherein thestimulation of an immune response in T cells is determined by measuringcellular markers of B cell activation.

In some aspects the immunogenic composition is contacted with an immunecell.

In some aspects the generation of an immunogenic composition comprisesgenerating structural ensembles, designing computation sequences,filtering the computation sequences, recombining designs, conformationalre-sampling of a representation of an interacting segment, and receivingan input related to human-guided design and filtering.

In some aspects the data storage is further configured to store at leastpart of the antigen variant library.

In some aspects the methods of the disclosure are performed using phage,yeast, or mammalian display.

In some aspects a cell of the disclosure is selected from the groupconsisting of: a bacterial cell, fungal cell, insect cell, animal cell,human cell and plant cell.

In some aspects the immunogenic composition elicits the production ofantibodies.

In some aspects the immunogenic composition elicits an adaptive immuneresponse, humoral immune response or an innate immune response.

In some aspects the immunogenic composition is directed towards anantigen related to one member selected from the following groupconsisting of: virus, Influenza virus, HIV virus, bacteria, parasite,fungus, infectious disease, autoimmune disease, inflammatory disease,neurological disease, addiction, cardiovascular disease, endocrinedisease and cancer.

In some aspects the library of a plurality of antigen variants is atleast 2, 1×10¹; 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹,1×10¹⁹, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, 1×10¹⁸,1×10¹⁹, 1×10²⁰, 1×10²¹, 1×10²², 1×10²³, 1×10²⁴, or 1×10²⁵ antigenvariants.

In some aspects the target epitope shares at least 75%, 80%, 85%, 90%,95%, 99%, 99.9% or 100% homology with target epitopes across a pluralityof antigen variants in the antigen library.

In some aspects the non target epitope regions shares at most 50%, 60%,65%, 70%, 75%, 80%, 85%, 90%, homology with non target epitope regionsacross a plurality of antigen variants in the antigen library.

In some aspects the antigen is from a chemical weapon or an agent of biowarfare.

In some aspects the antigen is a protein or peptide, lipoprotein, lipid,carbohydrate, nucleic acid or combination thereof.

In some aspects the immunogenic composition comprises a pharmaceuticallyacceptable carrier, excipient, adjuvant, or vehicle.

In some aspects the immunogenic composition comprises a chemotherapeuticagent, a targeting moiety, an anti-cancer agent, an adjuvant, or ahapten.

In some aspects the immunogenic composition comprises an enzyme.

In some aspects the antibody is a humanized monoclonal antibody.

In some aspects the target epitope is a ligand binding site, functionalmotif or enzyme active site.

In some aspects the immunogenic composition is selected from the groupconsisting of: enzyme, vaccine, pharmaceutical and therapeutic.

In some aspects, the immunogenic composition further comprises an agentselected from the following group consisting of: B-cell targetingmoiety, T-cell targeting moiety, anti-viral agent, chemotherapeuticagent, a toxin, immunostimulatory agent, adjuvant, and hapten.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of a device of this disclosure are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages of this disclosure will be obtained by referenceto the following detailed description that sets forth illustrativecases, in which the principles of a device of this disclosure areutilized, and the accompanying drawings.

FIG. 1 is a schematic representing 3 processes used for generating animmunogenic composition of the disclosure.

FIG. 2 is a schematic representing various in silico methods forgenerating a immunogenic composition of the disclosure.

FIG. 3 is a reference structure of the HA protein with various conserved(black) and non conserved (grey) regions of surface exposed epitopes onthe HA protein.

FIG. 4 is a schematic representation of storage and transmission of dataof the compositions and methods of this disclosure.

FIG. 5 is a schematic representation of 3 epitopes of the same targetprotein, and varying sequence identities of the 3 epitopes betweenantigens variants of the target protein.

DETAILED DESCRIPTION OF THE DISCLOSURE I. General Overview

The present disclosure provides compositions and methods for thegeneration of an antibody or immunogenic composition, such as a vaccine,through epitope focusing by variable effective antigen surfaceconcentration. Generally, the composition and methods of the disclosurecomprise three processes: a “design process”, (100), comprising one ormore in silico bioinformatics steps to select and generate a library ofpotential antigens for use in the immunogenic composition; a“formulation process”, (101), comprising in vitro testing of potentialantigens, using various biochemical assays, and further combining two ormore antigens to generate one or more immunogenic compositions; and an“administering process”, (102), whereby the immunogenic composition isadministered to immune cells, present either in vitro (i.e. cellculture) or in vivo (i.e. a host animal, subject or patient) as show inFIG. 1. Further steps may also be included, such as the isolation andscalable production of antibodies raised by host immune response to theimmunogenic composition.

In the design process, a suite of bioinformatics tools or algorithms arefirst used to analyze and compare protein structures of an antigen ofinterest. A plurality of antigen structures, each representing differentprotein conformations or structural variations (e.g. resulting frommutations or genetic variability) may be compared. An algorithm may beused to generate a map of the protein surfaces of a particular antigen.In some cases, the map may differentiate between areas on the proteinsurface that are conserved and non-conserved across the plurality ofantigen structures compared. In some cases, another algorithmic tool maybe used to select one or more epitopes from regions identified asconserved in the antigen map.

After one or more epitopes are selected (i.e. target epitopes), anadditional algorithm may be used to generate an in silico library ofantigenic variants, each variant comprising one or more conserved targetepitopes surrounded by non-conserved diversified surfaces. A subsequentalgorithmic step is applied, whereby antigenic variants generated in theprevious step may be filtered, according to various criteria, andselected to be combined in a mixture, or “ensemble,” to formulate theimmunogenic composition. In the formulation of the immunogeniccomposition, each antigenic variant may be assigned a specificconcentration in the ensemble such that the ensemble itself isimmunogenic, but that the concentration of individual antigenic variantsis not immunogenic, or insufficient to produce an immune response whencontacted with an immune cell.

In the formulation process, antigenic variants for one or more ensemblesmay be expressed and tested biochemically for various factors, such asprotein stability and epitope specificity. Individual antigenic variantsmay be combined in an ensemble, with each variant at a specificconcentration as predicted in the previous design process.

After biochemical testing and production of selected antigenic variants,an immunogenic composition is formulated from an ensemble of antigenicvariants and subsequently administered to a subject or immune cell. Insome cases, the subject may be a human subject in need of vaccination.In some cases, the subject or immune cell may be used to isolate anantibody raised against the immunogenic composition.

The compositions and methods may be particularly useful in variety ofmedical and biotechnology applications. For example, the compositionsand methods of the disclosure may be used to develop vaccines directedtowards the prevention and/or treatment of a variety of human diseases,ranging from infectious diseases from microorganisms such as bacteriaand viruses, to cancer. In other applications, the compositions andmethods of the disclosure may be used for the generation of antibodies,directed to a single epitope of interest in a protein. The generation ofspecific antibodies to a single epitope may have many applications,including the development of various antibody based tools and therapiesfor the identification, detection, diagnosis or treatment of variousdiseases. Further, compositions and methods of the disclosure may bealso used to generate antibody based tools for basic research functions,including but not limited to immunoassays, protein purification, proteinidentification, protein quantification, protein characterization, andprotein structural domain mapping.

II. Definitions

The terminology of the present disclosure is for the purpose ofdescribing particular cases only and is not intended to be limiting ofcompositions, methods and devices of this disclosure.

The compositions and methods of this disclosure as described herein mayemploy, unless otherwise indicated, conventional techniques anddescriptions of molecular biology (including recombinant techniques),cell biology, biochemistry, microarray and sequencing technology, whichare within the skill of those who practice in the art. Such conventionaltechniques include polymer array synthesis, hybridization and ligationof oligonucleotides, sequencing of oligonucleotides, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,equivalent conventional procedures can, of course, also be used. Suchconventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds.,Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler,Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNAMicroarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics:Sequence and Genome Analysis (2004); Sambrook and Russell, CondensedProtocols from Molecular Cloning: A Laboratory Manual (2006); andSambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (allfrom Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4thEd.) W.H. Freeman, N.Y. (1995); Gait, “Oligonucleotide Synthesis: APractical Approach” IRL Press, London (1984); Nelson and Cox, Lehninger,Principles of Biochemistry, 3rd Ed., W.H. Freeman Pub., New York (2000);and Berg et al., Biochemistry, 5th Ed., W.H. Freeman Pub., New York(2002), all of which are herein incorporated by reference in theirentirety for all purposes. Before the present compositions, researchtools and methods are described, it is to be understood that thisdisclosure is not limited to the specific methods, compositions, targetsand uses described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to limit thescope of the present disclosure, which will be limited only by appendedclaims.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another case includes from the one particular value and/or tothe other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another case. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. The term “about” as used herein refers to a range that is 15%plus or minus from a stated numerical value within the context of theparticular usage. For example, about 10 would include a range from 8.5to 11.5. The term “about” also accounts for typical error or imprecisionin measurement of values.

As used herein, the term “antibody” is meant to refer to immunoglobulinmolecules (e.g., any type, including IgG, IgE, IgM, IgD, IgA and IgY,and/or any class, including, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)isolated from nature or prepared by recombinant means or chemicallysynthesized. The terms “antibody” and “immunoglobulin” can be usedinterchangeably throughout the specification, unless indicatedotherwise.

As used herein, antibody may also refer to an antibody fragment whichmay represent a portion of a whole antibody which retains the ability toexhibit antigen binding activity or immunogenicity. Antibody andantibody fragment may be used interchangeably herein. Examples ofantibodies or antibody fragments include, but are not limited to, Fv,disulphide-linked Fv, single-chain Fv, Fab, variable heavy region (VH),variable light region (VL), and fragments of any of the above antibodyfragments which retain the ability to exhibit antigen binding activity,e.g., a fragment of the variable heavy region VH retains its ability tobind its antigen.

As used herein, the term antibody that “specifically (or selectively)binds to” or is “specific for” or is “specifically (or selectively)immunoreactive with” a particular polypeptide or an epitope on aparticular polypeptide is one that binds to that particular polypeptideor epitope on a particular polypeptide without substantially binding toany other polypeptide or polypeptide epitope. Antibody affinity forantigens can be measured by enzyme linked immunosorbent assay (ELISA).

Alternatively, an antibody that specifically binds to an antigen, mayrefer to the binding of an antigen by an antibody or fragment thereofwith a dissociation constant (Kd) of at least 3 μM, 2 μM, 1 μM, 900 nM,800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 1 nM,900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM,1 pM, 900 fM, 800 fM, 700 fM, 600 fM, 500 fM, 400 fM, 300 fM, 200 fM,100 fM, or 1 fM. In some cases antibodies or fragments thereof may havea dissociation constant (Kd) of at most 3 μM, 2 μM, 1 μM, 900 nM, 800nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 1 nM, 900pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 1pM, 900 fM, 800 fM, 700 fM, 600 fM, 500 fM, 400 fM, 300 fM, 200 fM, 100fM, or 1 fM as measured by any suitable biochemical assay, including butnot limited to surface plasmon resonance analysis using, for example, aBIACORE surface plasmon resonance system and BIACORE kinetic evaluationsoftware.

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler and Milstein, Nature256:495497 (1975); Kozbor et al, Immunology Today 4:72 (1983); Cole etal, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. (1985)). Techniques for the production of single chain antibodies(U.S. Pat. No. 4,946,778) can be adapted to produce antibodies topolypeptides of this disclosure. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al, Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

The term “immunoassay” is meant to refer to an assay that uses anantibody to specifically bind an antigen. The immunoassay ischaracterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the antigen.

As used herein, the terms “biological sample” or “patient sample” asused herein, is meant to refer to a sample obtained from an organism orfrom components (e.g., cells) of an organism. The sample can be of anybiological tissue or fluid. The sample may be a clinical sample which isa sample derived from a patient. Such samples include, but are notlimited to, sputum, blood, serum, plasma, blood cells (e.g., whitecells), tissue samples, biopsy samples, urine, peritoneal fluid, andpleural fluid, saliva, semen, breast exudate, cerebrospinal fluid,tears, mucous, lymph, cytosols, ascites, amniotic fluid, bladder washes,and bronchioalveolar lavages or cells therefrom, among other body fluidsamples. The patient samples may be fresh or frozen, and may be treated,e.g. with heparin, citrate, or EDTA, or other suitable treatment knownin the art. Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes.

As used in this disclosure, the term “epitope” is meant to refer to anyantigenic determinant on an immunogen, e.g., any primary immunogen, towhich an antibody binds through an antigenic binding site. Determinantsor antigenic determinants on an antigen usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics. In somecases, an epitope may be an area of surface exposed residues and/orcarbohydrate moieties on an antigen. In some cases, the area ranges from100 Å-1500 Å. In some cases, an epitope may be defined as a an area ofof surface exposed residues on an antigen ranging from at least 25 Å²,50 Å², 100 Å2, 200 Å², 300 Å², 400 Å², 500 Å², 600 Å², 700 Å², 800 Å²,900 Å², 1000 Å², 1100 Å², 1200 Å², 1300 Å², 1400 Å² or 1500 Å². In somecases, an epitope may be defined as a an area of of surface exposedresidues on an antigen ranging from at most 25 Å², 50 Å², 100 Å², 200Å², 300 Å², 400 Å², 500 Å², 600 Å², 700 Å², 800 Å², 900 Å², 1000 Å²,1100 Å², 1200 Å², 1300 Å², 1400 Å² or 1500 Å². In some cases, an epitopemay be 650-900. In some cases, an an epitope may range from 650 Å²-750Å², 750 Å²-800 Å², 800 Å²-850 Å² or 850 Å²-900 Å².

A “hapten” is a small molecule that, when attached to a larger carriersuch as a protein, can elicit the production of antibodies that bindspecifically to it (in either the free or combined state). A “hapten” isable to bind a preformed antibody, but fails to stimulate antibodygeneration on its own. In the context of this invention, the term“hapten” includes modified amino acids, either naturally occurring ornon-naturally occurring. Thus, for example, the term “hapten” includesnaturally occurring modified amino acids such as phosphotyrosine,phosphothreonine, phosphoserine, or sulphated residues such as sulphatedtyrosine (sulphotyrosine), sulphated serine (sulphoserine), or sulphatedthreonine (sulphothreonine); and also may include non-naturallyoccurring modified amino acids such as p-nitro-phenylalanine.

A “hapten-labeled antigen” in the context of this invention refers to ahapten attached to an antigen of interest. The antigen of interest maybe a peptide, or protein. The hapten can be positioned anywhere in theepitope of interest.

The terms “isolated,” “purified” or “biologically pure” may refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity may be typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation may be substantially purified. The term“purified” may denote that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Particularly, it mayindicate that the nucleic acid or protein is at least 85% pure, at least95% pure, or at least 99% pure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol Chem. 260:2605-2608 (1985);Rossolini et al., MoI. Cell. Probes 8:91-98 (1994)). The term nucleicacid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes. Aminoacid may also be referred to as “amino acid residue” or “residue,” usedinterchangeably herein.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. The probes can be directly labeled as with isotopes,chromophores, lumiphores, chromogens, or indirectly labeled such as withbiotin to which a streptavidin complex may later bind. By assaying forthe presence or absence of the probe, one can detect the presence orabsence of the select sequence or subsequence.

The term “recombinant” when used with a reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed and an operably linked to a promoter.

An “immune cell” is any immune cell capable of providing an immuneresponse to immunogenic stimuli, such as exposure to one or immunogensor ensemble of antigens. Immune cells may include but are not limited toNeutrophils, Eosinophils, Basophils, Lymphocytes, T cells, B cells,Cytotoxic, Plasma cells, T cells, Granulocytes, Helper T cells,Macrophages, Mast cells, Memory cells, Monocytes, platelets, Dendriticcells, antigen presenting cells (APCs), or any cell considered part ofan immune system of a host animal or subject. In some cases, an immunecell may also comprise a hybrid of one or more cells. For example, animmune cell may comprise a hybridoma, whereby an immune cell may befused to an immortalized cell, such as a cancer cell.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, may refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same. Insome cases, 2 or more sequences may be homologous if they share at least20%, 25%, 30%. 35%, 40%, 45% 50%, 55%, 60% identity, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identityto a reference sequence when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. In some cases, 2 or moresequences 2 or more sequences may be homologous if they share at most20%, 25%, 30%. 35%, 40%, 45% 50%, 55%, 60% identity, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identityto a reference sequence. This definition also refers to the complimentof a test sequence. Preferably, the identity exists over a region thatis at least 25 amino acids or nucleotides in length or in some casesover a region that is 50-100 amino acids or nucleotides in length. Insome cases, 2 or more sequences may be homologous and share at least 30%identity over at least 80 amino acids in a sequence according to theSander-Schneider homology limit.

Alternatively, an indication that two nucleic acid sequences orpolypeptides are identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two molecules or theircomplements hybridize to each other under stringent conditions, asdescribed herein.

For sequence comparison, generally one sequence acts as a referencesequence, to which test sequences may be compared. When using a sequencecomparison algorithm, test and reference sequences may be entered into acomputer, subsequent coordinates may be designated, if necessary, andsequence algorithm program parameters may be designated. Any suitablealgorithm may be used, including but not limited to Smith-Watermanalignment algorithm, Viterbi, Bayesians, Hidden Markov and the like.Default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm may then be used tocalculate the percent sequence identities for the test sequencesrelative to the reference sequence, based on the program parameters. Anysuitable algorithm may be used, whereby a percent identity iscalculated. Some programs for example, calculate percent identity as thenumber of aligned positions that identical residues, divided by thetotal number of aligned positions.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous or non-contiguous positions whichmay range from 10 to 600 positions. In some cases the comparison windowmay comprise at least 10, 20, 50, 100, 200, 300, 400, 500, or 600positions. In some cases the comparison window may comprise at most 10,20, 50, 100, 200, 300, 400, 500, or 600 positions. In some cases thecomparison window may comprise at least 50 to 200 positions, or at least100 to at least 150 positions in which a sequence may be compared to areference sequence of the same number of contiguous or non continguouspositions after the two sequences are optimally aligned. Methods ofalignment of sequences for comparison are well-known in the art. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman and Wunsch, J.MoI. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology (Ausubelet al, eds. 1995 supplement)).

In some cases, a comparison window may comprise any subset of the totalalignment, either contiguous positions in primary sequence, adjacentpositions in tertiary space but discontinuous in the primary sequence,or any other subset of 1 up to all residues in the alignment.

As used herein, the term “vaccine” is meant to encompass any immunogeniccomposition that is capable of inducing an immune response in a subject.The vaccine can include one or more immunogenic composition, e.g., anyprimary immunogenic composition together with a secondary immunogeniccomposition. Immune response may include responses that result in atleast some level of immunity in the subject to which the immunogeniccomposition is administered.

As used herein, the term “immunogenic composition” is any substance,formulation or organism that provokes an immune response (producesimmunity) when contacted with an immune cell. In some cases, immunogeniccompositions encompass all substances or formulations that can berecognized by the adaptive, humoral or innate immune system whenadministered to a host animal or subject. In some cases, immunogeniccompositions are those substances that elicit a response from the immunesystem.

As used herein, the term “intermediate antibodies” define antibodies(including B cell associated antibodies, i.e., BCRs) with zero tointermediate somatic mutational diversification on the maturationalpathway of an antibody from a germline antibody to a maturated antibody.An intermediate antibody can have zero or more mutated amino acidresidues compared to the germline antibody but has fewer mutatedresidues compared to the mature antibody. Analysis of somatichypermutations can be performed by classifying to the closest V-gene andJ-gene by VDJFasta algorithm or any other sequence classifier, aligningthe sequence to its germline counterparts using the VDJFasta algorithmor any other sequence alignment algorithm, and then taking the percentidentity of the alignment. The intermediate antibody may have between 0%to 95%, of the mutations of the corresponding mature antibody. In somecases the intermediate antibody has at least 0%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, or 90% of the mutations of the corresponding matureantibody. In some cases the intermediate antibody has at most 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, or 90% of the mutations of thecorresponding mature antibody.

The term “in vitro translation system”, which is used hereininterchangeably with the term “cell-free translation system” refers to atranslation system which is a cell-free extract containing at least theminimum elements necessary for translation of an RNA molecule into aprotein. An in vitro translation system may comprise at least ribosomes,tR As, initiator methionyl-tRNA^(Met), proteins or complexes involved intranslation, e.g., eIF2; eIF3, the cap-binding (CB) complex, comprisingthe cap-binding protein (CBP) and eukaryotic initiation factor 4F(eIF4F).

The term “somatic mutational diversification (SMD)” is a measure of thenumber of mutated amino acid residues compared to the germline and is aconsequence of the natural B cell diversification processes, includingaffinity maturational processes in which the B cell undergoeshypermutation in a germinal center in the presence of an antigen. It isexpressed as the percentage of that number compared to the total numberof amino acid residues in the sequences. Generally, the number of aminoacids encoded by the VH gene is used to measure the SMD because usuallythe heavy chain variable region is a major determinant of the antibodyspecificity and the VH gene encodes most of the amino acid residues ofthe heavy chain variable region. Sometimes, the VL gene can also be usedto analyze SMD.

III. Immunogenic Composition Design: Epitope Selection and Generation ofAntigenic Variant Ensembles A. Antigen Structural Analysis

The composition and methods of the disclosure first provide for a designprocess involving the structural analysis, (200) of one or more targetantigen proteins as shown in FIG. 2. Generally, a three dimensionalstructure, structural representative or structural model is firstobtained of a target antigen and analyzed for potential epitopes againstwhich an antibody may be raised. In some case, a target epitope may bechosen based on the presence of known or predicted neutralizingantibodies that may selectively bind to the target epitope. In somecases, an antigen may be a single protein of interest or a complex oftwo or more proteins. In some cases an antigen may refer to an antigendomain, or a region or section of a larger protein or protein complex.In some cases an antigen may be a fragment of a protein or a polypeptidesequence. Generally, an antigen selected for structural analysis may beany polypeptide sequence or glycoprotein.

An antigen can be an intact (i.e., an entire or whole) antigen, or afunctional portion of an antigen that comprises one or more epitopes. Anantigen may be a peptide functional portion of an antigen. “Intact”refers to full length antigen as that antigen polypeptide occurs innature. An intact antigen may adopt the native protein fold as seen innature, presenting both primary sequence epitopes as well as tertiarysequence conformational epitopes. This is in direct contrast to deliveryof only a small portion or peptide of the antigen. Delivering an intactantigen to a cell may elicit an immune response to a full range ofepitopes of the intact antigen, rather than just a single or selectedfew peptide epitopes.

Alternatively, an intact antigen can be divided into many parts,depending on the size of the initial antigen. Typically, where a wholeantigen is a multimer polypeptide, the whole protein can be divided intosub-units and/or domains where each individual subunit or domain of theantigen can be associated with the polymer according to the methods asdisclosed herein. Alternatively, in some cases, an intact antigen can bedivided into functional fragments, or parts, of the whole antigen, forexample, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95 Or 100 portions (e.g., pieces or fragments), inclusive,and where each individual functional fragment of the antigen can beassociated with the polymer according to the methods as disclosedherein. In some cases the antigen may be divided into at most 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 Or 100portions (e.g., pieces or fragments), inclusive.

The fragmentation or division of a full length antigen polypeptide canbe an equal division of the full length antigen polypeptide, oralternatively, in some cases, the fragmentation may be asymmetrical orunequal. Any combination of overlapping fragments of a full length wholeantigen may be used in the generation of subsequent antigen library asdescribed herein.

In some cases, an antigen may be selected for structural analysis basedon the availability of one or more three dimensional structures ormodels available for the protein. In some cases, structures orstructural models may not be available for a particular antigen.Available structures may include any suitable structures, as solved by avariety of techniques known in the art, including but not limited toX-ray crystallography (i.e. crystal structure), nuclear magneticresonance (NMR), small angle X-ray diffraction (SAX), cryo-electronmicroscopy, electron microscopy or in silico modeling methods, such asthreading or homology modeling. Generally, one or more proteinstructures may be obtained via any suitable database, including but notlimited to databases such as the publicly available Protein Data Bank(PDB) or the Molecular Modeling Database (MMDB). Structures could alsobe generated using any other known techniques in the art.

In some cases, an experimental protein structure may not be available.In some cases a structural model or representative may be used. If theexact crystal structure of a particular antigen is not known, but itsprotein sequence is similar or homologous to another known sequence witha known crystal structure, a homology model may be generated and usedwith the compositions and methods of this disclosure. Structural modelscan be generated using Modeller (N. Eswar, M. A. Marti-Renom, B. Webb,M. S. Madhusudhan, D. Eramian, M. Shen, U. Pieper, A. Sali. ComparativeProtein Structure Modeling With MODELLER. Current Protocols inBioinformatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6.30,200). Appropriate structural modeling templates can be identified bycreating a profile Hidden Markov Model (HMM) of the reference sequencesusing HMMer (Profile Hidden Markov Models. S. R. Eddy. Bioinformatics,14:755-763, 1998), and then using the HMM to search for homologousscaffolds in PDB with a minimum expectation value (e-value) of at1×10⁻³. In some cases, the e-value may be at least, 1×10⁻¹, 1×10²,1×10⁻³, 1×10⁻⁴, 1×10⁻⁵, 1×10⁻⁶. In some cases, the e-value may be atmost, 1×10⁻¹, 1×10⁻², 1×10⁻³, 1×10⁴, 1×10⁵, 1×10⁻⁶. In such instances,it is expected that the conformation of the particular antigen may besimilar to the known crystal structure of a homologous protein. Theknown structure may, therefore, be used as the reference structure forall variants of the target antigen, or in some cases, may be used topredict the structure of the target antigen (i.e., in “homologymodeling” or “molecular modeling”).

The resolution of protein structures for a selected antigen may rangefrom 1 Å to 100 Å. In some cases, protein structures for a selectedantigen may range from 1 Å to 6 Å. In some cases protein structures fora selected antigen may range from 1 Å to 3 Å, 2 Å to 4 Å, 3 Å to 6 Å, or4 Å to 6 Å. In some cases, a protein structure or structural model maybe at least 1 Å, 2 Å, 3 Å, 4 Å, 5 Å, 6 Å, 7 Å, 8 Å, 9 Å, 10 Å, 15 Å, 20Å, 25 Å, 30 Å, 35 Å, 40 Å, 45 Å, 50 Å, 60 Å, 70 Å, 80 Å, 90 Å, or 100 Å.In some cases a protein structure or structural model may be at most 1Å, 2 Å, 3 Å, 4 Å, 5 Å, 6 Å, 7 Å, 8 Å, 9 Å, 10 Å, 15 Å, 20 Å, 25 Å, 30 Å,35 Å, 40 Å, 45 Å, 50 Å, 60 Å, 70 Å, 80 Å, 90 Å, or 100 Å.

One or more antigen proteins may also be selected based on otherfactors, such as size, shape, complexity, ability to elicit broadlyneutralizing antibodies, biological relevance for an immunogeniccomposition or the degree of characterization as known in the art. Inyet other cases, an antigen may be selected based on known antibodiesthat may bind one or more epitopes on the protein.

For example, an antigen may range from 5 KD-1000 KD in size, as singleprotein or a complex of proteins. In some cases, an antigen may be atleast 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900 or 1000 KD in size. In some cases, an antigenmay be at most 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900 or 1000 KD in size.

For example, an antigen may range from 50 amino acids to-100,000 aminoacids in size, as a single protein or a complex of proteins. In somecases, an antigen may be at least 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000,80000, 90000, or 100000 amino acids in size. In some cases, an antigenmay be at most 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or100000 amino acids in size. In some cases, an antigen is at least 100amino acids in length. In some cases an antigen is a minimal number ofamino acids comprising a protein fold.

In the case of biological relevance for vaccine for example, an antigenmay be selected based on the amount of antigen expressed by a particularpathogen, or how well the antigen may be exposed to immune cells in ahost animal. Examples may include but are not limited to viral capsidproteins, surface receptors of cancer cells or parasites, transemembraneproteins of pathogens, secreted toxins, soluble growth factors and thelike. Generally any protein may be selected as an antigen.

Suitable antigen proteins may include but are not limited to ligands,cell surface receptors, cytokines, hormones, transcription factors,signaling molecules, cytoskeletal proteins, virulence factors, viralproteins, bacterial proteins, proteins from pathogens and enzymes.Suitable classes of enzymes include, but are not limited to, hydrolasessuch as proteases, carbohydrases, lipases; isomerases such as racemases,epimerases, tautomerases, or mutases; transferases, kinases,oxidoreductases, and phophatases. Suitable enzymes may be listed in theSwiss-Prot enzyme database. Suitable antigens may include, but are notlimited to, all of those found in the protein data base compiled andserviced by the Research Collaboratory for Structural Bioinformatics(RCSB, formerly the Brookhaven National Lab).

In some cases, an antigen may be selected, such that the antigen is notexpressed by a known pathogen. For example, in applications relating tothe development of antibody based tools, any protein may be designatedan antigen, whereby an antibody raised against an epitope on the proteinis desired.

After one or more protein structures or structural models are obtained,the structure is analyzed using any suitable algorithm that may be usedto estimate geometric relationships, such as distance, betweenindividual residues in the antigen. In the three dimensional folding ofa protein, amino acid residues, not found adjacent sequentially in theprimary sequence may be proximal to one another spatially. Generally, asuitable algorithm provides for estimating the geometric relationship,such as distance between residues defined by Cartesian coordinates. Forexample, one such distance geometry program, DGEOM, is a distancegeometry program for molecular model-building and conformationalanalysis available from Chiron Corporation of Emeryville, Calif. Havel,et al. J Theor Biol. 104:359-81 (1983); Havel et al. J Theor Biol.104:383-400 (1983). Another such tool is Modeller (N. Eswar, M. A.Marti-Renom, B. Webb, M. S. Madhusudhan, D. Eramian, M. Shen, U. Pieper,A. Sali. Comparative Protein Structure Modeling With MODELLER. CurrentProtocols in Bioinformatics, John Wiley & Sons, Inc., Supplement 15,5.6.1-5.6.30, 200), in which both structural proximity as well aspercent surface accessibility can be calculated. Molecular structurescan be described by the set of all pairs of interatomic distancesproduced using physical constraints of the protein structure. Distancemeasures between residues can also be obtained directly from the PDBfile by reading it using BioPerl, BioPython, or any other bioinformaticsframework.

For example, to identify all residues in a hypothetical epitope in a HAprotein centered around residue S100, a PDB model of HA may be accessedusing the bioinformatic program BioPerl in which a list of every residuewith a carbon alpha backbone position less than 25 Angstroms from thecarbon alpha backbone position of S100 is generated.

Further, antigen structural analysis may also provide informationrelating to which residues of the protein may be more surface exposedthan others, some of which may be buried in the interior of the protein.In some cases, certain algorithms may provide a score for the relative“exposure” of each residue in a protein structure or model. This can beperformed with Modeller (N. Eswar, M. A. Marti-Renom, B. Webb, M. S.Madhusudhan, D. Eramian, M. Shen, U. Pieper, A. Sali. ComparativeProtein Structure Modeling With MODELLER. Current Protocols inBioinformatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6.30,200).

In some cases, certain residues are readily exposed to the outsideenvironment. In some cases, certain residues are partially buried in thethree dimensional folds of the protein. In other cases, certainresidues, such as highly hydrophobic amino acids, are completely buriedin the interior core of the protein. Given that immunogenic B-cellepitopes are generally comprised of residues that are readily availableor exposed to a an immune receptor B cell receptor (BCR), residues thatare identified as highly exposed to the exterior of the protein andfound proximal one another, may be selected as part of one or morepotential epitopes of the antigen in subsequent steps (C. Epitopemapping) and described further herein. In other cases, MHC-dependentepitopes displayed to T cell receptor (TCR) may not be found in surfaceexposed regions and can be found anywhere in the protein. In some cases,epitopes may be derived from region of the protein that are not surfaceexposed.

B. Antigen Homolog Alignment

Generally, after initial antigen structural analysis, one or moresequences, homologous to the primary sequence of the target antigen maybe aligned and compared, (201). This step may be used to improveselection of functional variation of the protein, as determined bysubsequent steps (Surface Heterogeneity Optimization). A variety ofcomputational methods and strategies may be employed to align sequencesand compare three dimensional structures of the proteins sequences. Insome cases, protein structures are available for variety of homologousantigens or antigen variants. For example, for certain antigens such asHA protein or the HIV gp120 protein, numerous crystal structures havebeen solved for various mutation substitutions or variants, and many(e.g. thousands) of sequences are available in public databases, such aspublic influenza databases. In some cases these variants have beenexperimentally constructed (e.g. selected residues of recombinantprotein have been purposely mutated and the structure of the mutatedprotein solved), while in other cases these variants may exist in nature(e.g as a result of genetic variation in different strains of a virus).In this step of the composition and methods of the disclosure, anyprotein structures or models of relevant homologs may be aligned andcompared. In some cases, each homolog may contribute additionalinformation to variation observed to be tolerated on the native fold,and to identify variation never observed. In some cases, variationderived from natural variation may be used to identify amino acidpositions suitable for further diverisification or amino acids positionsthat do not tolerate variation and and may be avoided duringdiversification in subsequent steps described herein.

In some cases, sequences that may be homologous to the target antigen ortarget antigen domain with a known or predicted structure may share atleast 25%, 30%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.9%, 99.99% or 99.999% identity as described herein. Insome cases, the sequences homologous to the target antigen or targetantigen domain with a known or predicted structure share at most 25%,30%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 99.9%, 99.99% or 99.999% identity. Percent identity may bedetermined or calculated using a variety of computational techniques asdescribed herein. For example, homology can be calculated using the NCBIstandard BLASTp programs for protein using default conditions, inregions aligned together (without insertions or deletions in either ofthe two sequences being compared) including all residues for thesequences compared.

In certain cases, insertions or deletions or “gapped” sequences may bedisregarded. For example, the Thermus Aquaticus Phenylalanyl tRNASynthetase alpha subunit appears to have an “insert” region fromresidues 156 to 165 when compared to its homologs from other species.This region can be disregarded in calculating sequence identity.

Percent identity may be calculating using any suitable methods,including but not limited to creating a profile Hidden Markov Model of areference sequence using HMMer (Profile Hidden Markov Models. S. R.Eddy. Bioinformatics, 14:755-763, 1998), and then using the HMM tosearch for homologous scaffolds in PDB for homology with a minimumexpectation (e-value) or threshold. In some cases, the e-value may be atleast, 1×10⁻¹, 1×10⁻², 1×10⁻³, 1×10⁻⁵, or 1×10⁻⁶. In some cases, thee-value may be at most, 1×10⁻¹, 1×10⁻², 1×10⁻³, 1×10⁻⁴, 1×10⁻⁵, or1×10⁻⁶. Other programs that may provide similar calcuations includeMUSCLE, or mafft.

Alternatively, in some cases, the compositions and methods of thedisclosure also provide for comparison of homologous domains ofparticular proteins. For example, while the overall percent identity oftwo or more proteins may be less than 50%, certain regions of theproteins, such as one or more domains, might be used for sequencealignment. In this case, an epitope may be selected from a desireddomain homologous to the proteins and sequences aligned and compared.

Generally, antigen homologs may be compared from known antigen variants,or by variants generated or predicted computationally. In some cases,mutations or variations made to the target antigen sequence may yield aprotein that does not have a structure solved by experimentation. Inmany cases, a variety of techniques may be used to model the predictedstructure of these variants or homologs. In some cases, modeledstructures may use a reference. In some cases modeled structures may bereference-free.

Generally, any of the many methods of model generation can be applied atthis step in the over-all methodology. The alignment methods describedherein are provided only for example. Other methods may be used todeduce structures that are consistent with distance and general modelconstraints. Two strategies that may be particularly useful with thecompositions and methods of the disclosure are constrained threading andconstrained sequence/structure alignment. Other possible methods includebut are not limited to dynamic programming and clique detection.

For example, constrained threading is a general computational strategyto generate models by threading a sequence through a database ofsequence-unique protein folds. Various software programs are availablein the art to generate such models. For example, the publicallyavailable algorithm provided by Alexandrov et al. “Fast Protein FoldRecognition via Sequence to Structure Alignment and Contact CapacityPotentials.” Protein Science Bulletin. (1996), incorporated by referenceherein, may be used. This program involves entering the sequence of theprotein, determining the alignment mode and allowing the softwarealgorithm to generate the model. In global alignments, all positions areconsidered. In free shift alignments gaps at the beginning or at the endare not scored. Local alignments may be maximal common substringalignments. For any of these alignment modes, the program may provide agiven number of top scoring alignments.

Various models may be generated for a single homolog and evaluated usingvarious constraints and parameters. Parameters such as total constrainterror, the number of distance constraints, the pairwise distanceseparation, and the pairwise distance as defined by the structure orreference for the two residues in a constraint may be used to evaluatethe most useful models.

Other programs or algorithms may be used in homolog alignment. In somecases, one or more algorithms may use an overall stepped process such asthreading. In this case, the primary sequence of the protein beinganalyzed is threaded through each selected protein structure. Forexample, the backbone of the antigen homolog under consideration is laidon top of a backbone for the currently selected target antigen. Afterthe homolog under consideration has been aligned with a selected proteinsuch as the structure of the target antigen, the selected protein isscored. In some cases a score based on (1) sequence identity between thetwo proteins, (2) alignment of secondary structures between the twoproteins, and (3) a contact capacity potential of the protein in itsthreaded format. The second scoring criterion may involve approximatingsecondary structures of the protein based on the primary sequence. Thethird scoring criterion may be based on the how closely the localenvironment (neighboring amino acids) of an amino acid residue matcheswith its empirically-determined preferred environment. Other softwareprograms and other scoring criteria (e.g., hydrophobicity, potentialmean force) can be used.

Residues of selected models may be subsequently converted into threedimensional coordinates by a computer program such as DGEOM, asdescribed herein, and compared to the target antigen structure.

Another computational strategy for antigen homolog alignment maycomprise constrained sequence/structure alignment. This approach employsthe constraints to first build a set of structural models, and thenevaluate models by applying a pairwise hydrophobic contact potential toeach model, and rank-ordering models based on this potential function.An algorithm as provided by Bryant et al. “An Empirical Energy Functionfor Threading Protein Sequence Through the Folding Motif,” Proteins.1993 16 92-112, incorporated by reference in its entirety herein, may beused. In this approach, alignments to the fold may be defined bysystematically matching residues of the target protein or antigen linkedby a restraint to residues of the fold for which the interatomicdistance of the alpha carbons is less than the extended crosslinker plusside chain atoms (<23.85 Angstroms in the case of the BS3 linkers).

The protein sequence can then be mapped onto the fold working back fromthe first-matched residue to the first residue of the sequence, or tothe first of the fold, forward from-the first matched residue and backfrom the second in a symmetrical fashion, and forward from the secondmatched residue. Alignments can be scored using the pairwise hydrophobiccontact potential defined by Bryant et al., 1993, and the best scoreobtained for each fold was retained to rank the fold.

In both model generation approaches, the model most complementary toknown constraints and/or experimental parameters may be selected for theconstruction of a homology model. The threading alignment can be used tomatch amino acids in the sequence to positions in the structure. Otheralignment protocols could be used as well. The model can then beconstructed using standard homology modeling techniques. Additionally,distance constraint violations within the model may assist in furtherrefinement of the model. Refinement of the model can be done usingdistance geometry, energy minimization, and/or molecular dynamics.

Any of the techniques, or combination of techniques as described hereinmay be used to generated predicted structural models for antigen homologalignment.

In some cases, master-slave modeling may be used in alignment ofstructures, whereby a pivot (master) structure may be aligned to allother structures (slaves) to it based on pairwise alignments. If thereis any error or inconsistency in the master-slave alignments, the finalmultiple structural superposition and alignment may be erroneous. Onesuch program, CE-MC (Guda et al., 2001) may be used whereby a subsequentMonte Carlo step to increase the number of aligned columns and correcterrors. Another widely used technique is the progressive approach (PAL)(Yang and Honig, 2000; Ye and Godzik, 2005 and may be used with thecompositions and methods of this disclosure. Positions in the model maybe then mapped the alignment for all members and then to the profile ofthe hiden markov model coordinates of the design.

As described herein, structural models generated computationally, orsynthetically, may be performed for a range of homologs. In some cases,1-1,000,000 homologs may be generated or obtained and structurallyaligned to the target antigen. In some cases at least 1, 10, 100, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000,400000, 500000, 600000, 700000, 800000, 900000, or 1000000 differenthomologous structures of antigen variants may be computationallygenerated or obtained and compared to the target antigen. In some casesat most 1, 10, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000,100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000,or 1000000 different homologous structures of antigen variants may becomputationally generated or obtained and compared to the targetantigen. All or part of the collection of homologs generated may becompared for further analysis.

C. Epitope Identification and Selection

After one or more homologous antigen structures have been generated orobtained, multiple structures may be compared spatially to identify andselect a target epitope on the target antigen, (202). In some cases, atarget epitope is a patch of structurally proximal, surface exposedresidues on the antigen, such as for an epitope for BCRs. In some cases,an epitope is a patch of adjacent residues found anywhere in theprotein, such as in surface exposed, or non surface exposed regions ofthe antigen. Non surface exposed epitopes may be used to select epitopesfor T-cell receptors (TCRs). In some cases, when a known antibody, suchas a neutralizing antibody, has been previously characterized, one ormore target epitopes on an antigen surface may refer to all uniquecombinations of residues where the maximum solvent exposed surfacecircumference distance in A between any two members is less than orequal to the maximum distance between an antigen residues observed inknown antibody-antigen contacts from published structures. In this case,information regarding the specific spatial binding position of anantibody to a target epitope may be determined experimentally or bepublically available. Co-crystallization, high resolution cryo-EM, orNMR of an antibody or antibody fragment bound to a target epitope on anantigen may provide this experimental information. Antibody epitopes mayrange from 100 Å² to 1500 Å² as described herein.

Alternatively, information for a given target epitope and antibody maynot be known. In some cases, a target epitope may refer to allcombinations observed by in-silico protein-protein docking of multipleantibody models against the target epitope on the target antigen. Insome cases, a target epitope across two homologs of the antigen proteinmay be scored as function of target epitope percent identity. Percentidentity may be calculated as the following: Target Epitope PercentIdentity=((Residues in Target Epitope)−(Hamming Distance between TargetEpitopes in Homologs))/(Residues in Target Epitope). In some case TargetEpitope average similarity may be calculated as the following: TargetEpitope average similarity=Σ (BLOSUM62 similiarity score between allhomologous positions)/Residues in Epitope).

In some cases, no previous known information regarding antibody-epitopebinding interactions may be previously known, whereby in silico dockingmay be limited or unable to be performed. In some cases, a targetepitope may be selected de novo from the structural comparisons ofsequence homologs. In some cases, structural comparison of variants mayprovide information regarding the identification of conserved surfaceexposed residues in an antigen. Antigens, for which there are numerousvariants found in nature, such as viral proteins, may be particularlyuseful. In other cases, in silico model generation may provide somepredicative information regarding surface residues that must remainconserved for stability of the protein. In some cases, conserved surfaceresidues of a particular antigen may be determined experimentally, or asknown in the art. A protein map comprising multiple aligned structuresof one or more homologs and the target antigen may be generated. Astructural comparison map may be useful for determining which residuesmay comprise an epitope (a related by geometric distances) and which ofthese epitopes may be most highly conserved.

In one example, this strategy may be particularly useful in the designof a functional vaccine. In the example of viral protein such as HAprotein, the influenza virus may be able to mutate to evade multipleantibodies generated to epitopes that are prone to mutation. Slightchanges to the genome of the virus translate to differences in theresidues of the protein. These changes may cause the virus to escapeimmune recognition. This step of the composition and methods of thedisclosure may allow for the identification of surfaces of “broadlyneutralizing epitopes” in the antigen protein that may be resistant tosuch changes. Identification and ultimate selection of these areas fortarget epitopes to which an antibody may be raised, may decrease thelikelihood of viral evasion of antibodies through mutation or antigenicdrift in a host or subject. Epitope selection and strategies for vaccinedevelopment are further described herein.

D. Surface Heterogeneity Optimization

After a target epitope is identified and/or selected, another algorithmis used to generate a population or library of antigenic variantsthat 1) maintain the three dimensional conformation and spatialspecificity of the target epitope, while 2) varying the sequencecomposition and spatial specificity of the protein surface surroundingthe target epitope, (203). The methods and compositions of thedisclosure provide for the generation of an immunogenic compositioncomprising two or more antigen variants, whereby the effectiveconcentration of a target epitope common to all antigen variants ishigher than the concentration of any additional surface features orregions of the antigen surrounding the target epitope. In effect, thisstep may help to diversify the antigen regions surrounding the targetepitope such that the diversity decreases the effective concentration ofnon-target epitope surface. This strategy may produce a focused immuneresponse to the desired target epitope, while avoiding an immuneresponse to undesired regions of the antigen (i.e. non target epitopesurfaces of the protein).

The compositions and methods of the disclosure provide for a generalcomputational strategy for surface heterogeneity optimization ofmultiple antigenic variants or members. In some cases, an “inverseprotein folding” strategy may be used for optimization of amino acidsequences in non-target epitope surfaces of the protein. Similar toprotein design, such approach seeks to find a sequence or set ofsequences that will fold into a desired structure. Antigenic members,each containing a conserved target epitope, may still fold in a similarfashion as the target antigen. In some cases similar folds between thetarget antigen and antigenic members is desired. In some cases, thesimiliarity of folds between antigenic variants and the target antigenmay aid in the correct presentation of the native target epitope. Unlikealternative approaches in vaccine development, whereby target epitopes,such as a peptide or protein domain may be isolated out of context ofthe full protein as a lineage peptide or fused to additional sequences,the target epitope is conserved across a plurality of antigenic membersand may be exposed to the exterior of the protein in a conformationsimilar to the conformation as found in the original target antigen. Insome cases, presentation of the epitope in conformations similar to theepitope's native conformation may increase the likelihood of strong andtargeted immune response to the natural or native target epitope.

In a generalized approach, non target epitope residue positions that areselected for heterogeneity optimization may be determined based variouscriteria. Information related to relative sequence conservation orpredicted protein stability models may indicate which residues may beeasier to change and optimize than others. During optimization andgeneration of antigen variants, changes in sequence in non-targetepitope regions may be selected to have minimal impact on theconformation of the target epitope region of the antigen and the generalfolds of the antigen. In some cases, residues that have the least impactmay be more prone for selection and change during the optimizationprocess. In some cases, changes to a combination of residues may have animpact or little impact on the target epitope region. The generalstrategy of the algorithm is optimization of maximal change to nontarget epitope regions of the antigen variants, wherein the antigenvariant still adopts a similar protein fold of the target antigen, andthe target epitope remains spatially conserved between the antigenvariant and the original target antigen. In some cases, maximal changeis reflected by maximal decrease in precent identity of comparable nontarget epitope regions between antigenic variants.

In some cases, a set of homologs may be diversified in the non targetepitope regions by first generating a positional weight matrix for allpositions in the antigen sequence. As described herein, antigen homologsequences may be first selected and aligned, such as with the programMUSCLE. Probability scores from the alignment may then be used togenerate a probabilistic, statistical or stochastic model for subsequentanalysis of individual amino acid positions in the sequnce. Generally,any suitable model may be used. For example, hidden markov, dynamicprogramming, support vector machine, Bayesian network, trellis decoding,Viterbi decoding, expectation maximization, Kalman filtering, or neuralnetwork methodologies may be used. Various stuiable programs may be usedto generate these models. For example, as described herein, the programHMMer may be used to generate a hidden markov model (HMM). The HMM maybe used to search any database, such as PDB for homolog structures ormodels. One or more strucutural models may then be aligned using the HMMderived for each sequence.

After the structural models have been aligned, a positional weightmatrix (PWM) may be extracted from the HMM. Generally a positionalweight is a matrix of score values that gives a weighted match to anygiven substring of fixed length. It has one row for each symbol of thealphabet, and one column for each position in the sequence. The scoreassigned by a PWM to a substring s=(s_(j))_(j=1) ^(N) is defined Σ_(j=1)^(N)m_(s) _(j) _(,j), where j represents position in the substring,s_(j) is the symbol at position j in the substring, and m_(α,j) is thescore in row α, column j of the matrix. In some cases, a PWM score isthe sum of position-specific scores for each symbol in the substring. Insome cases, the PWM can be interpreted as the identity and frequency ofamino acids permitted at each structurally conserved position in allknown variants of the antigen.

Using a PWM, each position in the structure may be mapped to a positionin the alignment, correlating a column in the HMM, and a column in thePWM. Further, amino acids identified as surface exposed, as describedherein, may be mapped to each column in the PWM from the structure (e.gas provided by a program such as Modeller).

Further, identified epitopes, as described herein may be mapped to thePWM. In some cases, epitopes are known in the art for a particularantigen or protein. In some cases, an epitope is chosen for a particularantigen or protein as described herein. In some cases, non-exposedresidues may be assigned as reference sequence and may be excluded fromdiversivication. In some cases, non surface exposed residues or epitopesmay be computationally masked to prevent diversification. With thistrategy, non exposed residues, or residues found in the core of theprotein may be conserved, while exposed residues outside the targetepitope region are diverisified.

Diversification of non-target epitope surface exposed sequences may beperformed by manipulating diversity frequencies in the remainder of thepositions that are surface exposed and not part of target epitopes.Using computation-guided simulations, individual amino acid positionsmay be diversified by substituting a different amino acid residue foreach position, in silico. Diversification of each position provides forthe generation of sequences that may then be further tested andanalyzed.

For example, a collection of 1×10⁶ sequences can be sampled from the PWMdesign in-silico by bioinformatics simulation and analyzed. If thesequences are either so similar to one another that they contain manyoff-target epitopes in common, or so distant from the referencestructure that they have a low probability of folding, the PWM may bealtered and retested to generate additional sequences.

Optimization or altering of the PWM may be performed by techniques suchas linear scaling of non-dominant amino acid frequencies compared todominant amino acid frequencies at each position. Testing of moleculesmay comprise observing identity across one or more antigens. In somecases, antigen variant molecules may be at least 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical identicalto a known reference sequence. In some cases, antigen variant moleculesmay be at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, or 90% identical. In some cases, percent identity of oneantigen to another may reflect homology. In some cases, percent identityof one antigen to another may reflect the likelihood of antigensequences generated from the PWM to maintain the protein fold of thereference sequence. Generally any percent identity between antigens maybe accepted, provided the protein fold of the antigen is conserved inone or more antigen variants generated by the PWM for the library.

Further, testing comprises observing percent identity of a plurality ofnon target epitopes, defined as any collection of surface exposedresidues within a defined area centered on the carbon alpha backbone ofa residue also on the surface. In some cases, the area is at least 25Å², 50 Å², 100 Å², 200 Å², 300 Å², 400 Å² or 500 Å², 600 Å², 700 Å², 800Å², 900 Å², 1000 Å², 1200 Å², 1500 Å², or 2000 Å². In some cases, thearea is at most 25 Å², 50 Å², 100 Å², 200 Å², 300 Å², 400 Å² or 500 Å²,600 Å², 700 Å², 800 Å², 900 Å², 1000 Å², 1200 Å², 1500 Å², or 2000 Å².In some cases, the epitope area is 25-100 Å². In some cases, the epitopearea is 100-200 Å². In some cases, the epitope area is 200-300 Å². Insome cases, the epitope area is 300-400 Å². In some cases, the epitopearea is 400-500 Å².

Generally, the precent identity of non target epitopes may be comparedpairwise between antigen variants generated by the PWM. For example, acertain residue position may be chosen in the antigen and one or moreantigen variants. A residue may also be chosen between different antigenvariants. Percent identity of all residues within a defined area, suchas 25 Å² around the chosen residue, may be compared and percent identitycalculated. The non target epitope area may be described by any suitablegeometric shape, including but not limited to an oval, disc, circle,square, triangle, rectangle, star, polygon and the like. In some cases,the area is defined by an oval shape.

The precent identities of a plurality of non target epitope areas may becompared in a pairwise analysis. In some cases percent identity greaterthan 90% of a commonly defined area between antigen variants mayindicate the antigen variants may be too similar in those non targetepitope regions. In some cases, non target epitope regions or areas maybe at at least 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%identical. In some cases, non target epitope regions or areas may be atat most 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% identical.

For example, for a given protein as shown in FIG. 5, multiple epitopesare found on the surface of a target antigen. In this example, 3epitopes are shown, epitope A, (501), epitope B, (502), and epitope C,(503) for different antigen variants. In this example, epitope B ischosen as the target epitope, while epitopes A and C are chosen fordiversification. As described herein, diversification yields changes inpercent identity of non-target epitopes across antigen variants of animmunogenic composition. Using a commonly defined oval area of 100 Å²,percent identity may be calcuated for each epitope (A, B, C) across eachof the antigen variants. The commonly defined area may be centeredaround a particular residue within the epitope or area adjacent to theepitope. In the case of epitope A, a non target epitope region, theaverage percent identity is determined to be 62% in this example.Similiarly, for epitope C, another non epitope region, the averagepercent identity is determined to be 66% in this example. For the targetepitope region, epitope B, the epitope is common to all antigen variantsand has an average percent identity of 95%. Given the relativedifference between the percent identity of epitope B and epitopes A andC, epitope B is considered conserved between the antigen variants.

Further, the cartoon diagrams as shown in FIG. 5, indicates a similifiedmodel of BCR recognition of epitopes A, B and C. When combined together,the 3 antigen variants of this example immunogenic composition presentdifferent effective concentrations of epitopes A, B and C. Given thediversification of epitopes A and C across the 3 epitopes, BCRrecognition of either epitope A (504) or recognition of epitope C (506)is relatively low, as indicated by binding of only one molecule byeither B cell. However, the effective concentration of epitope B, due toits conservation (high precent identity) across all antigen variants inthe immunogenic composition is higher than for A or C. Thus BCRrecognition may be higher for epitope B, (505) as indiciated by thebinding of multiple antigens by the B cell. This differentialrecognition of epitopes based on differences in effective concentrationprovides for eliciting an immune response selectively to the targetepitope.

In some cases, if sequence generated by the PWM fails to meet athreshold as described herein, the PWM may be altered to be either moreor less diverse. As an alternative to PWM-library optimization, a humanuser could manually design and test variant sequences.

As an alternative to linear scaling, a random monte carlo stochasticsampling, a genetic algorithm, or manual or human user interventioncould also be used to optimize the final PWM.

In some cases for selected residues in the non target epitope regions,the side chains of any positions to be varied are then removed. Theresulting structure consisting of the protein backbone (including thefixed backbone structure of the analog) and the remaining sidechains maybe called the template. Each variable residue position can then beoptionally classified as a core residue, a surface residue, or aboundary residue. In that case, each classification defines a subset ofpossible amino acid residues for the position (for example, coreresidues generally will be selected from the set of hydrophobicresidues, surface residues generally will be selected from thehydrophilic residues, and boundary residues may be either). Each aminoacid residue or the analog can be represented by a discrete set of allallowed conformers of each side chain, called rotamers. Thus, to arriveat different potential antigenic variants for a backbone, all possiblesequences may be screened, where each backbone position can be occupiedeither by each amino acid in all its possible rotameric states, or asubset of amino acids. For this purpose, the analog backbone is treatedas part of the target antigen template.

In some cases, not all possible sequences may be screened for eachbackbone position. In some cases, the frequency of amino acids andindividual positions may be used to determine which positions may bemore tolerant than others for diverisification. In some cases, wherethere is homolog data available, amino acid position frequency may beused to provide further skewing opmitization in the PWM, whereinpositions that are tolerant of changes may be diversified, whilepositions that are evolutionarily convserved may be masked or blockedfrom diversification.

For sequences that meet percent identity thresholds, (e.g. exhibitnon-target precent identity less than 90% and target epitopeconservation), sequence may be further tested in silico for predictedbiochemical stability. For example, a criterion such as theoreticalquantitative stability may be used as a measure of the stability of aconformation and thus stability of the presented conserved targetepitope. For example, different antigen sequences may be tested bycalculating ΔG of individual folded molecules. Molecules for which ΔG issubstantially higher than the reference sequence or antigen may beexcluded or lower ranked (as described herein). In other silico tests,stability may be tested by applying a thermal function to predictedfolds of individual antigen variants. Variants that are found to bethermally unstable (e.g. predictd to denature, unfold, or improperlyfold), may be excluded or lower ranked in the library.

Methods to rank rank-list sequences may performed using any suitablemethods. For example profile scores (Bowie et al., Science253(5016):164-70 (1991), incorporated by reference) and/or potentials ofmean force (Hendlich et al., J. Mol. Biol. 216(1):167-180 (1990), alsoincorporated by reference) can also be calculated to score sequences.These methods assess the match between a sequence and a 3D proteinstructure and hence can act to screen for fidelity to the proteinstructure. By using different scoring functions to rank sequences,different regions of sequence space can be sampled in the computationalscreen.

Furthermore, scoring functions can be used to screen for sequences thatwould create metal or co-factor binding sites in the protein (Hellinga,Fold Des. 3(1):R1-8 (1998), hereby expressly incorporated by reference).Similarly, scoring functions can be used to screen for sequences thatwould create disulfide bonds in the protein. These potentials attempt tospecifically modify a protein structure to introduce a new structuralmotif.

Similarly, molecular dynamics calculations can be used tocomputationally screen sequences by individually calculating mutantsequence scores and compiling a rank ordered list.

In a preferred embodiment, residue pair potentials can be used to scoresequences (Miyazawa et al., Macromolecules 18(3):534-552 (1985),expressly incorporated by reference) during computational screening.

Similarly, as outlined above, other computational methods are known,including, but not limited to, sequence profiling (Bowie and Eisenberg,Science 253(5016): 164-70, (1991)), rotamer library selections (Dahiyatand Mayo, Protein Sci 5(5): 895-903 (1996); Dahiyat and Mayo, Science278(5335): 82-7 (1997); Desjarlais and Handel, Protein Science 4:2006-2018 (1995); Harbury PNAS USA 92(18): 8408-8412 (1995); Kono etal., Proteins: Structure, Function and Genetics 19: 244-255 (1994);Hellinga and Richards, PNAS USA 91: 5803-5807 (1994)); and residue pairpotentials (Jones, Protein Science 3: 567-574, (1994)), all of which areexpressly incorporated by reference.

The computational processing results in a set of optimized proteinsequences that each contain the target epitope displayed in a nativeconformation as the target antigen, and non target epitope sequencesthat have been optimized to fold in a similar fashion as the targetantigen, but comprise a protein surface highly divergent from surfacesof the target antigen. These optimized protein sequences may besignificantly different from the wild-type sequence from which thebackbone was taken. That is, each optimized protein sequence maycomprises at least one residue change, or at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11,%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,80%, or 90% or more variant amino acids from the starting or wild-typesequence. In some cases each optimized protein sequence may comprises atmost one residue change, or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11,%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, or 90% ormore variant amino acids from the starting or wild-type sequence.

The library or a population of optimized sequences, may be outputted asa rank-ordered list. Generally, all possible sequences of a protein maybe ranked; however, the number of sequences may be capped due tocomputational limitation. Thus, in general, some subset of all possiblesequences may be used as the primary library. In some cases a librarymay include at least 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷,1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶,1×10¹⁷, 1×10¹⁸, 1×10¹⁹, 1×10²⁰, 1×10²¹, 1×10²², 1×10²³, 1×10²⁴, 1×10²⁵,1×10³⁰, 1×10³⁵, 1×10⁴⁰, or 1×10⁵⁰ antigen variants or antigen members.In some cases a library may include at most 1×10¹, 1×10², 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹ 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, 1×10¹⁸, 1×10¹⁹, 1×10²⁰, 1×10²¹, 1×10²²,1×10²³, 1×10²⁴, 1×10²⁵, 1×10³⁰, 1×10³⁵, 1×10⁴⁰, or 1×10⁵⁰ antigenvariants or antigen members. Generally, the top or highest ranked 1×10³to 1×10¹³ sequences are chosen for the library. The cutoff for inclusionin the rank ordered list of the primary library can be done in a varietyof ways. For example, the cutoff may be just an arbitrary exclusionpoint: the top 1% of sequences may comprise the library. Alternatively,all sequences scoring within a certain limit of the global optimum canbe used; for example, all sequences with 10 kcal/mol of the globaloptimum could be used as the primary library. This method has theadvantage of using a direct measure of fidelity to a three dimensionalstructure to determine inclusion. This approach can be used to insurethat library mutations are not limited to positions that have the lowestenergy gap between different mutations. Alternatively, the cutoff may beenforced when a predetermined number of mutations per position isreached. As a rank ordered sequence list is lengthened and the libraryis enlarged, more mutations per position are defined. Alternatively, thetotal number of sequences defined by the recombination of all mutationscan be used as a cutoff criterion for the primary sequence library. Insome cases the values for the total number of sequences range from 100to 1×10²⁰. In some cases values range from 1000 to 1×10¹³. In some casesvalues range from 1000 to 10×10⁷. Alternatively, the first occurrence inthe list of predefined undesirable residues can be used as a cutoffcriterion. For example, the first hydrophilic residue occurring in acore position would limit the list. It should also be noted that whilethese methods are described in conjunction with limiting the size of theprimary library, these same techniques may be used to formulate thecutoff for inclusion in the secondary library as well.

E. Variable Surface Concentration Ensemble Generation

After generation of antigen variants in silico, another algorithm isused to aid in selection and formulation of an immunogenic compositioncomprising two or more antigen variants, (204). The composition andmethods of the disclosure provide for generation of one or moreimmunogenic compositions, wherein an immunogenic composition may be ableto elicit an immune response from a subject or immune cell, but thatindividual antigen variants or antigen members of the immunogeniccomposition may be held at a concentration insufficient to elicit animmune response. In some cases, the concentration of antigen members istoo low to elicit a response individually. However, when combined intoan ensemble for an immunogenic composition, the sum of the effectiveconcentration of the target epitope, as found across antigen members inthe ensemble, is sufficient to elicit an immune response. The algorithmused, to generate a variable surface concentration ensemble (or mixtureof antigen members or antigen variants) employs a general strategy tocombine different antigen variants into a mixture, wherein there issufficient separation between effective concentrations of the targetepitope and non-target epitopes that an immunization can be expected toonly result in sufficient concentration of antigen for B-cell or T-cellactivation of the target epitopes, but not the non target epitopessurfaces.

In some cases, each antigen member or antigen variant is assigned ascore, based on the predicted concentration of the target epitope. Theconcentration of the target epitope may be calculated as a function ofsimilarity (e.g. percent identity) across all target epitopes in theantigen member library generated in the previous step as well asdisimiliarity across non target epitopes (e.g. percent identity). Forexample, target epitopes that may be predicted to have undergone morespatial change than others may be assigned a lower concentration score.The algorithm may then select either a predetermined number or nonpredetermined number of antigen members or antigen variants to create anensemble mixture, and assign each antigen member or variant aconcentration, such that the concentration of target epitope equals orexceeds a certain threshold concentration. In some cases the thresholdconcentration may be the minimum concentration of target epitoperequired to elicit an immune response.

In some cases, an ensemble may include at least 2, 5, 10, 15, 20, 25,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000,50000, or 100000 antigen members or antigen variants. In some cases, anensemble may include at most 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, or 100000antigen members or antigen variants.

Additionally, in some cases, some antigen variants may not contain thetarget epitope. In some cases, a particular ensemble may comprise amixture of antigen variants. In some cases, at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 90%, 95%, 99%, 99.999% of an ensemble may compriseantigen variants without a target epitope. In some cases, at most 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 99.999% of an ensemblemay comprise antigen variants without a target epitope.

In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70 80, 90 or 100 ensembles may be generated for a particularantigen. In some cases, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70 80, 90 or 100 ensembles may be generated for a particularantigen.

IV. Formulation: Biochemical Testing and Formulation of an ImmunogenicComposition

After the design process, the various algorithms as described herein,provide one or more ensembles, each ensemble comprising a plurality ofantigenic members or variants designed and optimized in in silico.Generally, the design process provides amino acid sequences of all theantigenic variants in an ensemble. In order to develop and formulate theimmunogenic composition from the ensemble, recombinant protein for eachensemble may be made in vitro for the subsequent administering process,wherein the immunogenic composition is exposed to the immune system ofan animal or subject or an immune cell.

A. General Recombinant Methods for Expression of Antigen Protein

Antigen variants or antigen members of an ensemble may be produced invitro using a variety of methods including recombinant and traditionalbiochemical purification methods.

In some cases sequences as provided by the design process for variousantigen variants may be cloned into an appropriate expression vector andexpressed in a host cell. Successful protein expression and/orbiochemical testing of individual proteins may be used to validate thestability of the protein sequence and utility as a member of theimmunogenic composition to be formulated for administration.

Generally, after the design process, an ensemble, comprising a pluralityof antigen variant sequences, is generated in silico. The amino acidsequences generated in this step may be reverse translated intocorresponding nucleic acid sequences. Nucleic acid sequences may befurther optimized by applying codon optimization techniques known theart for particular expression systems to be used. For example, nucleicacid sequences may be codon optimized for expression in E. coli orinsect cells, or human cells or expression in virtually any host cell asdescribed herein.

Generally, any suitable host cell may be used to express antigen variantproteins. Generally, host cells may include bacteria, (e.g. E. coli),fungal cells, (e.g. S. Cerevisiae), insect cells (Sf9, Hi5, Sf21 celllines), animal cells (e.g. CHO, HEK293, HeLa), plant cells (N. tabacum,A. thaliana) and the like. Host cells may be present in vitro (i.e.tissue culture cells or cell lines), or in vivo (i.e. cells in anorganism).

As mentioned previously, methods well known to those skilled in the artmay be used to construct cloning vectors containing antigen variantsequences, transcriptional and translational control elements and DNAsequences. Exemplary techniques are described in Sambrook, J. et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y., and Green, E. etal. (1997) Genome Analysis, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y. In some cases, sequences generated by the designprocess may be synthetically manufactured prior to cloning intoexpression vectors.

In order to obtain antigen expression of sufficient quantity, cloning ofthe antigen sequences into an expression vector may be employed. Anexpression vector may contain necessary elements for transcriptionand/or translation of the inserted coding sequences. Expression vectorsand systems known in the art may be employed for producing full lengthor only portions of the polypeptides of the biological agents andcompounds of the disclosure.

Generally, nucleic acid sequences of individual antigen variants areoperably linked to one or more transcriptional control sequences, e.g.,a promoter and an enhancer. Generally, such nucleic acids are alsoincorporated into a plasmid or an expression vector, which is thenintroduced into a host cell to allow expression of the protein. The typeof transcriptional control sequences used may depend on the particularexpression system used, e.g., whether the system is prokaryotic (e.g.,bacterial) or eukaryotic (e.g., yeast, avian, insect or mammalian), oran in vitro transcription system.

In some cases, the expression system is a prokaryotic expression system.Generally, a nucleic acid encoding a protein of interest is operablylinked to one or more transcriptional control elements, such as apromoter; the nucleic acid is introduced into a prokaryotic host cell;and the host cell is cultured such as to produce the protein ofinterest. A plasmid may comprise sequences required for appropriatetranscription of the nucleic acid in bacteria, e.g., a promoter and atranscription termination signal. The vector or plasmid can furthercomprise sequences encoding factors allowing for the selection ofbacteria comprising the nucleic acid of interest, e.g., gene encoding aprotein providing resistance to an antibiotic and sequences required forthe amplification of the nucleic acid, e.g., a bacterial origin ofreplication. Exemplary vectors for the expression of a protein inprokaryotic cells, such as E. coli, include but are not limited toplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids.

Any of the numerous prokaryotic expression systems known in the art canbe used in the invention. Numerous systems are commercially available,(e.g., from Novagen and Life Technologies). Exemplary systems aredescribed herein. The expression vector can be introduced into theprokaryotic host cells according to methods known in the art, e.g., heatshock transfection of chemically competent cells or electroporation.Host cells having incorporated the expression vector are then identifiedand used for the production of the protein of interest.

The nucleic acid encoding the protein of interest can be under thecontrol of an inducible promoter. Such promoters are well known in theart and are found in commercially available vectors. The presence of aninducible promoter facilitates expression of proteins that may otherwisebe toxic to the host cells. For example, the powerful phage T5 promoter,which is recognized by E. coli RNA polymerase, can be used together witha lac operator repression module to provide tightly regulated, highlevel expression or recombinant proteins in E. coli. Such vectors areavailable commercially, e.g., from Qiagen (Chatsworth, Calif.;QIAexpress pQE vectors). Other inducible promoters are those that areinducible by iron or in iron-limiting conditions. In some cases, aninducible promoter is used which can be activated by temperature,isopropylthio-beta-galactoside (IPTG), NaCl, or other stimuli.

Eukaryotic protein expression systems can be based on any suitableeukaryotic species (e.g., mammalian cells, insect cells, yeast cells andplant cells). Generally, a nucleic acid encoding a protein of interestis operably linked to at least one transcriptional control element,e.g., a promoter and an enhancer. Eukaryotic transcriptional controlelements are well known in the art and are described, e.g., in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990).

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIPS, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein).

In addition, drug resistance markers such as ampicillin, zeomycin,bleomycin, DHFR, or neomycin can be used for selection of prokaryotic oreukaryotic host cells containing the recombinant vector.

An alternative eukaryotic expression system which can be used to expressa recombinant protein is an insect system. For example, a baculovirusexpression system can be used. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pNL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUWI), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

In another insect system, Autographa californica nuclear polyhedrosisvirus (AcNPN) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells (Sf cells). The gene sequence maybe cloned into non-essential regions (for example the polyhedrin gene)of the virus and placed under control of an AcNPN promoter (for examplethe polyhedrin promoter). Successful insertion of the coding sequencewill result in inactivation of the polyhedrin gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedrin gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed, (e.g., see Smith et al., 1983, J. Nirol., 46:584,Smith, U.S. Pat. No. 4,215,051).

In cases in which plant expression vectors are used, the expression of aprotein may be driven by any of a number of promoters and expressoisystems. For example, viral promoters such as the 35S RNA and 19S RNApromoters of CaMV (Brisson et al., 1984, Nature, 310:511-514), or thecoat protein promoter of TMN (Takamatsu et al., 1987, EMBO J.,6:307-311) may be used; alternatively, plant promoters such as the smallsubunit of RUBISCO (Coruzzi et al., 1994, EMBO J., 3:1671-1680; Broglieet al., 1984, Science, 224:838-843); or heat shock promoters, eg.,soybean lisp 17.5-E or hsp 17.3-B (Gurley et al., 1986, Mol. Cell.Biol., 6:559-565) may be used. These constructs can be introduced intoplant cells using Ti plasmids, Ri plasmids, plant virus vectors; directDNA transformation; microinjection, electroporation, etc. For reviews ofsuch techniques see, for example, Weissbach & Weissbach, 1988, Methodsfor Plant Molecular Biology, Academic Press, New York, Section VIII, pp.421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9.

In some cases, mammalian expression vectors may contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMN, pSN2gpt, pSN2neo,pSN2-dhfr, pTk2, pRSNneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare non-limiting examples of mammalian expression vectors suitable fortransfection of eukaryotic cells. Some of these vectors are modifiedwith sequences from bacterial plasmids, such as pBR322, to facilitatereplication and drug resistance selection in both prokaryotic andeukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPN-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and17.

In some cases, stable expression of polynucleotide sequences of eachantigen member sequence in a host cell line may be used. For example,cell lines which stably each antigen may be generated. An individualcell line for each antigen member may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells, which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

In some cases generating trangenic expression cell lines or organismscontaining stably integrated expression constructs may be useful forlong-term, high-yield production of recombinant antigen members orantigen variants.

In another example, a transgenic organism may be generated for theproduction of one or more antigen variants. For example, transgenicplants may be engineered to express one or more antigen variants, suchas A. thaliana or N. tabacum. In some cases, an appropriately engineeredvector comprising polynucleotide sequence of the antigen variant andvarious regulatory sequences (i.e. promoter sequence, terminatorsequences etc. . . . ) may be introduced into a plant via agrobacteriamediated “floral dip” transformation as known in the art. Transgenicseeds may be recovered, wherein stable integration of the antigenvariant sequence containing construct has occurred. Transgenic seeds maythen be propagated to produce plants expressing the antigen variants andsubsequently used to extract, purify and test the antigen variantprotein.

As another example, a host cell strain or organism may be chosen for itsability to modulate the expression of the inserted sequences or toprocess the expressed proteins or peptides in the desired fashion. Suchmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the protein may also be used to facilitate correct insertion,folding and/or function. Different host cells such as CHO, HeLa, MDCK,HEK293, and W138, which have specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the antigen.

In other cases, expression of antigen variant sequences may also begenerated using transient expression systems using any of the host,vectors, or methods described herein. For example, insect cells may usedto generate protein using transient expression of constructs usingbaculo virus as described herein. Plants may also be used for transientexpression of constructs using viral based methods, or agrobacteriamediated methods as described herein.

In some cases generating transgenic expression cell lines or organismscontaining transient expression constructs may be useful for generatingand testing antigen variant proteins faster and in high-throughputcompatible formats.

Generally, any suitable amount of protein may be expressed. In somecases, antigen variant protein may be produced in quantities of at least1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg,400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 ng, 10 ng, 100 ng, 200ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg,100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mgor 1 g. In some cases, antigen variant protein may be produced inquantities of at most 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg,300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg,700 mg, 800 mg, 900 mg or 1 g.

B. High Throughput Methods for Antigen Variant Expression

In some cases, such as in instances in which numerous antigen variantsof an ensemble may be produced, high throughput expression methods maybe used.

In some cases phage display may be used, whereby a plurality of antigenvariants or antigen member sequences of one or more ensembles arepooled. Using methods in the art, each antigen variant may be expressedon a portion of the phage, including areas such as capsid or tail. Phagedisplay methodologies, may be used to test antigens recognized byantibodies as provided by (Scott, J. K. et al. (1990) “Searching forPeptide Ligands using an Epitope Library,” Science 249(4967):386-390),incorporated by reference in its entirety herein.

In some cases a phage display library may be constructed to express oneor more antigen variants. In some cases, antigen variants are displayedon the surface of a phage in the form of a fusion with a coat protein ofthe phage. This chimeric outer surface protein is the processed productof the polypeptide expressed by a display gene, or antigen variantsequence, or partial antigen variant sequence, inserted into the phagegenome. Generally, the genome of the phage may allow introduction of thedisplay gene either by tolerating additional genetic material or byhaving replaceable genetic material; the virion may be capable ofpackaging the genome after accepting the insertion or substitution ofgenetic material; and the display of the coat protein-antigen variantprotein fusion on the phage surface may not disrupt virion structuresufficiently to interfere with phage propagation.

When the viral particle is assembled, its coat proteins may attachthemselves to the phage: a) from the cytoplasm, b) from the periplasm,or c) from within the lipid bilayer. The immediate expression product ofthe antigen variant may comprise, at its amino terminal, a functionalsecretion signal peptide, if the coat protein attaches to the phage fromthe periplasm or from within the lipid bilayer. If a secretion signal isnecessary for the display of the antigen variant protein, in some cases,the bacterial cell in which the antigen variant is expressed is of a“secretion-permissive” strain.

In some cases, the polynucleotide sequence encoding the antigen variantmay precede the sequence encoding the coat protein proper if the aminoterminal of the processed coat protein is normally free, or may followit if the carboxy terminal is the normal free end.

When variegation is introduced, multiple infections could generatehybrid viral particles that carry the gene for one antigen variant buthave at least some copies of a different antigen variant on theirsurfaces; in some cases, this may be minimized whereby cells areinfected with phage under conditions resulting in a lowmultiple-of-infection (MOI). For a given bacteriophage, a coat proteinis usually one that is present on the phage surface in the largestnumber of copies, as this allows the greatest flexibility in varying theratio of coat protein-antigen variant to wild type coat protein and alsogives the highest likelihood of obtaining satisfactory affinityseparation. One example of suitable coat protein may include but is notlimited to M13 gIII protein.

In many cases the wild-type coat protein gene is preserved. The antigenvariant sequence may be inserted either into a second copy of therecipient coat protein gene or into a novel engineered coat proteingene. In some cases the coat protein and antigen variant sequence areplaced under control of a regulated promoter including but not limitedto promoters such as lacUV5, tac, or trp.

Generally, any suitable position for insertion of the antigen variantgene into a phage genome may be used. In some cases bacteriophage arehighly ordered, such as a filamentous phage. Filamentous phage can bedescribed by a helical lattice; isometric phage, by an icosahedrallattice. Each monomer of each major coat protein sits on a lattice pointand makes defined interactions with each of its neighbors. Antigenvariants that fit into the lattice by making some, but not all, of thenormal lattice contacts may destabilize the virion by: a) abortingformation of the virion, b) making the virion unstable, or c) leavinggaps in the virion so that the nucleic acid is not protected. In somecases use phage display to produce antigen variants for ensemblesemploys techniques for engineered coat protein-antigen variant fusionproteins such that those residues of the parental coat proteins thatinteract with other proteins in the virion are kept intact in theassembled virion.

In some cases, such as M13 gVIII, the entire mature protein may beretained. In other cases, engineering a coat protein-antigen variantfusion protein capable of functional phage assembly may be performed bytruncating the coat protein. In some cases, at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the wildtype coatprotein may be used in a fusion with an antigen variant. In some cases,at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or100% of the wildtype coat protein may be used in a fusion with anantigen variant.

In some cases, the coat protein may be mutated or altered to engineer aa coat protein-antigen variant fusion protein capable of functionalphage assembly. In some cases at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99% or 100% of the wildtype coat protein may bemutated or altered in a fusion with an antigen variant. In some cases atmost 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%of the wildtype coat protein may be mutated or altered in a fusion withan antigen variant.

In some cases, the individual phage library may be used to express ordisplay an individual antigen variant (i.e. all phage display anidentical antigen variant). In other cases, a phage library may be usedto one or more antigen variants (i.e. phage display non-identicalantigen variant). In some cases, one more phage libraries may becombined to create an ensemble as part of a immunogenic composition.Further formulations of immunogenic compositions comprising phagelibraries are described herein.

Failed expression or expression of non functional protein may be quicklytested using such a method or any of the methods as described herein.

The recombinant antigen variant proteins can also be produced in an invitro system, e.g., in an in vitro transcription and translation system.Many vectors for in vitro transcription are available commercially.These may contain one or more of the promoters SP6, T3 and T7 and mayadditionally contain a poly A sequence at the 3′ end of the poly linkerin which the DNA of interest is inserted. Vectors that can be used forin vitro transcription are also described, e.g., in U.S. Pat. No.4,766,072. In vitro transcription can be conducted with a nucleic acidthat is not per se a vector, but merely contains the elements necessaryfor in vitro transcription. For example, such a template nucleic acidmay comprise an RNA polymerase promoter located upstream of the antigenvariant sequence to transcribe. Such template nucleic acids can beobtained, e.g., by polymerase chain reaction (PCR) amplification of asequence of interest using a primer that contains an RNA polymerasepromoter. PCR amplification methods are well known in the art.

An in vitro transcription reaction can be carried out according tomethods well known in the art. Kits for performing in vitrotranscription kits are also commercially available from severalmanufacturers.

In an illustrative case, a vector containing an RNA Polymerase promoterand an antigen variant sequence of interest is preferably firstlinearized downstream of the antigen variant sequence by e.g.,restriction digest with an appropriate restriction enzyme. Thelinearized DNA is then incubated in the presence of ribonucleotides, anRNAase inhibitor, an RNA polymerase recognizing the promoter that isoperably linked upstream of the insert to be transcribed. Following thetranscription reaction, RNAase free DNAse can be added to remove the DNAtemplate and the RNA can be purified by, e.g., a phenol-chlorophormextraction. Further details and variations of this general method may befound in the art (e.g., in Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989)).

In vitro synthesized RNA can be in vitro translated using an in vitrotranslation system. A variety of in vitro translation systems are wellknown in the art and are commercially available. Examples of in vitrotranslation systems include eukaryotic lysates, such as rabbitreticulocyte lysates, rabbit oocyte lysates, human cell lysates, insectcell lysates and wheat germ extracts. Lysates are commercially availablefrom manufacturers such as Promega Corp., Madison, Wis.; Stratagene, LaJolla, Calif; Amersham, Arlington Heights, 111; and GIBCO/BRL, GrandIsland, N.Y. In vitro translation systems typically comprisemacromolecules, such as enzymes, translation, initiation and elongationfactors, chemical reagents, and ribosomes.

Synthesis of recombinant proteins can be generally adapted to highthroughput, e.g., in multi-well plates. For example, proteins can beexpressed in multi-well plates using the Rapid Translation System ofRoche (RTS 100 E. coli HY Kit; Roche). This kit contains everythingneeded to perform protein expression in tubes or multi-well plates, andincludes E. coli lysate, reaction mix, amino acid mixture (withoutmethionine), methionine, reconstitution buffer, GFP control vector, and200 μl thin-walled tubes.

In certain high throughput embodiments, nucleic acids encoding proteinsof interest for use in an in vitro transcription and translation assay,such as that of the RTSIOO system from Roche, are prepared in amulti-well plate, in which it is then transcribed and translated. Forexample, a nucleic acid comprising a sequence encoding a protein ofinterest is incubated in a well of a multi-well plate together with twoprimers and reagents for conducting PCR.

For example, the amplified product can comprise the promoter at its 5′end, to permit in vitro transcription. After conducting a PCR reactionto amplify the nucleic acid encoding the protein of interest, andoptionally linking a promoter to it, the nucleic acid may be used in anin vitro transcription reaction. The method may comprise a step ofremoving certain reagents used in the PCR reaction prior to in vitrotranscription and translation. For example, one or both primers can beremoved from the reaction. Alternatively, the PCR product can bepurified away from some or most of the PCR reagents. For example, thePCR product can be synthesized with a label (which will essentially notaffect the transcription of the PCR product), e.g., biotin, and the PCRproducts isolated on an avidin or streptavidin solid surface, e.g.,beads.

In another example, high throughput protein expression may includemethods using two-hybrid systems, whereby a library of antigen sequencesare introduced in plurality of individual host cells and induced toexpress individual antigen variants. In some cases, this method, asknown in the art, may use yeast as the host cell. In other cases, insectcells or mammalian cells may be suitable for protein expression.

C. Protein Purification

Generally, after successful expression, any suitable means for proteinpurification may be used. Exemplary techniques are described inSambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, Plainview, N.Y., Ausubel, F. M. et al. (1989)Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., and Green, E. et al. (1997) Genome Analysis, A Laboratory Manual,Cold Spring Harbor Press, Plainview, N.Y.

Purifying an identified mutant protein comprises separating it(completely or partially) from at least one contaminant. Identifiedmolecules can be purified from undesired contaminants purified via oneor more purification steps. Some purification processes can result in a“homogeneous” preparation comprising at least 70% (e.g., at least 80%,at least 90% by weight, or at least 95%) by weight of the identifiedmolecule(s). Other purification processes (e.g., obtaining a celllysate, cell extract or cell culture supernatant) can result in a lowerdegree of purification, which may nonetheless be suitable for aparticular use. For example, cell lysates and cell extracts can be usedto make an in vitro translation transcription (IVTT) system.

Steps for purifying identified protein(s) from cultured cells can dependon whether the identified protein(s) remains inside cultured cells orare secreted into the cell culture growth medium. For identifiedproteins that remain within cultured cells, purification typicallyinvolves disrupting the cells (e.g., by mechanical shear, freeze/thaw,osmotic shock, chemical treatment, and/or enzymatic treatment). Suchdisruption results in a cell lysate that contains the identifiedmolecule and other cellular constituents. In some cases, much of theundesired cellular material can be removed by filtration orcentrifugation to yield a cell extract that contains the partiallypurified omolecule.

Chromatographic techniques often are used to further purify anidentified protein from cell culture growth medium, products of cellularmetabolism, and/or other cellular constituents. Such techniques canseparate polypeptides on the basis of size, charge, hydrophobicity, orpresence of purification tags, to name a few. Chromatographic separationschemes can be tailored to particular identified polypeptides, using oneor more chromatographic techniques and/or separation media. Duringchromatographic separation, an identified polypeptide can move at adifferent rate through a separation medium, or can adhere selectively tothe separation medium, relative to undesired molecules. In addition, anidentified protein can be positively selected or negatively selected.Thus, in some negative seletion schemes using chromatographicseparation, identified molecules can be separated from undesiredmolecules when the undesired molecules adhere to the separation mediumand the identified molecule(s) do(es) not. In such a scheme, theidentified molecules are present in the eluate or flow-through andundesired molecules are retained in association with the separationmedium. Alternatively, in positive selection schemes, the identifiedmolecules can be separated from undesired molecules when identified(desired) molecules adhere to the separation medium and undesiredmolecules do not. In such a scheme, the eluate or flow-through containsundesired molecules, and the separation medium retians the theidentified proteins. The identified molecules can be then be recovered,for example, by exposing the separation medium to a chemical orenzymatic agent suitable for dissociating the desired polypeptide.

Ion exchange chromatography is just one chromatographic technique thatcan be used to purify the identified proteins. In ion exchangechromatography, charged portions of molecules in solution are attractedby opposite charges of an ion exchange medium when the ionic strength ofthe solution is sufficiently low. Solutes can be dissociated from an ionexchange medium and eluted from an ion exchange column by increasing theionic strength of the solution. Changing the pH to alter solute chargeis another way to dissociate solutes from an ion exchange medium. Ionicstrength and/or pH can be changed gradually (gradient elution) orstepwise (stepwise elution).

Metal ion affinity chromatography (MIAC) is another chromatographictechnique that can be used to purify identified molecules. MIAC is anaffinity chromatography technique that involves the binding of desiredmolecules to metal ions. Immobilized metal ion affinity chromatography(IMAC) is a type of MIAC technique that involves the use of a separationmedium to which metal ions have been chelated. The identifiedpolypeptides may be immobilized on such a metal chelate substrate,reportedly via interaction(s) between metal ion(s) and electron-donatingamino acid(s) such as histidine and cysteine. Thus, IMAC routinely isused to purify recombinant polypeptides that include polyhistidine orpolycysteine motifs (tags). Whether, and with what affinity, aparticular desired polypeptide will bind to a metal chelate substratecan depend on the conformation of the polypeptide, the number ofavailable coordination sites on the chelated metal ion ligand, and thenumber of amino acid side chains available to bind the chelated metalion ligand.

The nature of a tag will depend on the particular affinity purificationsystem used. Various systems are available. In one embodiment, theaffinity chromatographic system is immobilized metal affinitychromatography (IMAC), which is based on binding of a tag to a metal ionresin. Metal ions can be, e.g., zinc, nickel, or cobalt ions. The tagcan be a polyhistidine sequence, which interacts specifically with metalions such as nickel, cobalt, iron, or zinc. A polyhistidine tag can be2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, or 50×His or other, provided that it binds essentiallyspecifically to a metal ion. In some cases, the His tag is a 6×His or12×His tag. The tag can also be a polylysine or polyarginine sequence,comprising at least four lysine or four arginine residues, respectively,which interact specifically with zinc, copper or a zinc finger protein.Commercially available systems for IMAC include the following systems,which are sold as kits and as individual components, e.g., vectors,bacterial strains, affinity resins and instructions for use: QIAexpressNi-NTA Protein Purification System of Qiagen (Qiagen, CA); HATT™ ProteinExpression & Purification System (Clontech, Palo Alta, Calif.); pTrcHisXpress™ Kit (InVitrogen); and BugBuster™ HisEind® Purification Kit(Novagen).

In a preferred embodiment, the invention comprises purifying a pluralityof recombinant proteins in multi-well plates. The affinity resins may bepresent on magnetic beads, thereby allowing easy removal of the beadsfrom the wells.

In some cases the tag peptide comprises a glutathione-S-transferase(GST) fusion protein and the affinity purification comprises usingglutathione, GST or an antibody to GST. Systems for expressing andpurifying recombinant proteins comprising a GST tag are available fromNovagen as BugBuster™ GST′Bind™ Purification Kit and GST-Tag™ Assay Kit.Exemplary vectors for producing such fusion proteins include the pGEXprokaryotic expression vectors from Pharmacia (Piscataway, N.J), e.g.,pGEX-5. GST fusion proteins can be affinity purified usingglutathione-Sepharose (Sigma Chem. Co.; St. Louis, Mo.) resin;GST-sepharose (Phamarcia-LKB); resin linked to an antibody specific forGST, e.g., mouse anti-GST-Sepharose® 4B (Zymed Laboratories). Proteinpurification can be performed as described, e.g., in Kuge et al. (1997)Protein Science 6: 1783.

Other affinity purification systems comprise a T7 tag, e.g., availablein the T7*Tag® Purification Kit (Novagen); an S tag or thioredoxin(trxA) tag (Novagen); and a Self

Cleavable Chitin-binding Tag, e.g., in the IMPACT™-TWIN System andIMPACT™-CN System (New England Biolabs); or a myc epitope or a peptideportion of the Haemophilus influenza hemagglutimn protein, against whichspecific antibodies can be prepared and also are commercially available.Other affinity systems include maltose sepharose or agarose affinitychromatrography using a maltose binding protein, and lectin affinitychromatography.

Additional affinity purification systems are based on the interactionbetween a tag peptide and an antibody to the tag peptide. Tag specificantibodies can be raised using a protein containing the tag peptide, ora peptide portion thereof, as an immunogen. Such an immunogen can beprepared from natural sources, produced recombinantly, or can besynthesized using routine chemical methods. An otherwise non-immunogenicepitope can be made immunogenic by coupling the hapten to a carriermolecule such bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH), or by expressing the epitope as a fusion protein. Various othercarrier molecules and methods for coupling a hapten to a carriermolecule are well known in the art (see, for example, Harlow and Lane,“Antibodies: A laboratory manual” (Cold Spring Harbor Laboratory Press1988)).

Electrophoresis techniques can also be used to purify desiredpolypeptides. Electrophoresis is based on the principle that chargedparticles migrate in an applied electrical field. If electrophoresis iscarried out in solution, molecules are separated according to theirsurface net charge density. If carried out in semisolid materials(gels), the matrix of the gel adds a sieving effect so that particlesmigrate according to both charge and size.

Gel-based electrophoresis can be carried out in a variety of formats,including in standard-sized gels, minigels, strips, gels designed foruse with microtiter plates and other high throughput (HTS) applications,and the like. Two commonly used media for gel electrophoresis and otherseparation techniques are agarose and polyacrylamide gels. In general,electrophoresis gels can be either in a slab gel or tube gel form.

Electrophoresis can performed in the presence of a charged detergentlike sodium dodecyl sulfate (SDS) which coats, and thus equalizes thecharges of most polypeptides such that migration is more dependent uponsize (molecular weight) than charge. Polypeptides often areelectrophoresed in the presence SDS, e.g., SDS-PAGE techniques. Inaddition to SDS, one or more other denaturing agents, such as urea, canbe used to minimize the effects of secondary and tertiary structure onthe electrophoretic mobility of polypeptides. Such additives typicallyare not necessary for nucleic acids, which have a similar surface chargeirrespective of their size and whose secondary structures generally arebroken up by the heating of the gel that happens during electrophoresis.

Isoelectric focusing (IEF) is an electrophoresis technique that involvespassing a mixture through a separation medium having a pH gradient orother pH function. An IEF system has an anode at a position ofrelatively low pH end and a cathode disposed at another position ofhigher pH. Molecules having a net positive charge under the acidicconditions near the anode will move away from the anode. As they movethrough the IEF system, molecules enter zones having less acidity,causing their positive charges to diminish. Each molecule will stopmoving when it reaches a point in the system having a pH equivalent toits isoelectric point (pI).

Two-dimensional (2D) electrophoresis involves a first electrophoreticseparation in a first dimension, followed by a second electrophoreticseparation in a second, transverse dimension. In a common 2Delectrophoretic method, polypeptides are subjected to IEF in apolyacrylamide gel in the first dimension, which results in separationon the basis of pI, and the molecules are then subjected to SDS-PAGE inthe second dimension, resulting in further separation on the basis ofsize.

Capillary electrophoresis (CE) achieves molecular separations on thesame basis as conventional electrophoretic methods, but does so withinthe environment of a narrow capillary tube (25 to 50.mu.m). The mainadvantages of CE are that very small volumes of sample are all that arerequired, and that separation can be performed very rapidly, thusincreasing sample throughput relative to other electrophoresis formats.Examples of CE include capillary electrophoresis isoelectric focusing(CE-IEF) and capillary zone electrophoresis (CZE). Capillary zoneelectrophoresis (CZE) is a technique that separates molecules on thebasis of differences in mass to charge ratios, which permits rapid andefficient separations of charged substances. In general, CZE involvesintroducing a sample into a capillary tube and applying an electricfield to the tube. The electric potential of the field pulls the samplethrough the tube and separates it into its constituent parts.Constituents of the sample having greater mobility travel through thecapillary tube faster than those with slower mobility. As a result, theconstituents of the sample are resolved into discrete zones in thecapillary tube during their migration through the tube. An on-linedetector can be used to continuously monitor the separation and providedata as to the various constituents based upon the discrete zones. Itshould be recognized that a recombinant protein fused to a tag peptideor other second polypeptide is in a sufficiently purified form to allowMS analysis, since the mass of the tag peptide will be known and can beconsidered in the determination. The tag peptide can also be cleavedfrom the polypeptide prior to the MS analysis, as described infra.

D. Biochemical Validation

In some instances, antigen variants or antigen members may be furthervalidated with various biochemical assays. Successful expression ofsoluble protein, as determined by the presence of protein expressionand/successful purification of protein may provide some informationregarding validation of the antigen variants. However furtherinformation may be required. For example, antigen members or variantsmay be tested for target epitope specificity using a variety ofbiochemical tests. In some cases, the protein structure may bedetermined, as with common methods in the art, such as X-raycrystallography. In other cases, biochemical assays using a knownbinding antibody may be used.

In cases in which an antibody may already be characterized or available,various immuno assays such as ELISA, western blot or surface Plasmonresonance may be used to assess the binding affinity for the targetepitope for a particular antibody. This information may be used tovalidate or reject predicted affinities from the design process and/ormay be used to adjust the “effective concentration assigned each antigenvariant or member.

In another assay, the binding of the target epitope to a T-cell orB-cell may be determined using other immuno based assays such asfluorescence activated cell sorting (FACS). In this case, a pool ofimmune cells may be exposed to a purified antigen, and relativepercentages or counts of cells with affinity for the antigen may beassessed and correlated with the strength of binding for the targetepitope.

In addition to activity or specificity based assays, general biochemicalstability and conformational specificity may also be assess using othertraditional biochemical techniques. For example, differential scanningcalorimetry (DCS) may be used to assess the melting temperature ofindividual antigen variants or members. This method may be used toassess the stability of the protein at various temperatures. Othertraditional techniques may include but are not limited to fluorescence,circular dichroism spectroscopy, hydrogen exchange-mass spectroscopy,differential scanning fluorometry (DSF), protein crystallization, massspec, MALDI-TOF and pulse proteolysis. Other methods may be used usingspecific ligands or binding agents that may be capable of detectingmisfolding protein or exposure of the hydrophobic core, such as the useof sypro orange. Generally any biochemical assay that providesinformation about either the conformational stability of antigenvariants, or specificity of recognition of an antibody to a targetepitope on the antigen variant may be used to validate each antigenvariant. Validated antigen members may be further selected to beincorporated into a formulation of an immunogenic composition.

Apart from this and/or in addition, the antigens selected forformulation of the immunogenic composition and the immunogeniccomposition itself may show only low to undetectable levels ofaggregation even during storage under one or more of the above stressconditions. For example, at least 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%,2%, 3%, 4%, 5%, 10%, 20% or 30% of the antigen variant or immunogeniccomposition are aggregated after storage under one or more of the abovestress conditions. In some cases at most 0.01%, 0.05%, 0.1%, 0.2%, 0.5%,1%, 2%, 3%, 4%, 5%, 10%, 20% or 30% of the antigen variant orimmunogenic composition are aggregated after storage under one or moreof the above stress conditions.

Aggregation as used in the present disclosure may include thedevelopment of high molecular weight aggregates, i.e. aggregates with anapparent molecular weight in SE-HPLC analysis of more/higher than the 44kDa. As described above, 44 kDa is the apparent molecular weightobserved in SE-HPLC analysis for dimers. Aggregation can be assessed byvarious methods known in the art. Without being limiting, examplesinclude SE-HPLC, analytical ultracentrifugation, dynamic lightscattering and/or subvisible particle counting.

In an analytical ultracentrifuge, a sample being spun can be monitoredin real time through an optical detection system, using ultravioletlight absorption and/or interference optical refractive index sensitivesystem. This allows the operator to observe the evolution of the sampleconcentration versus the axis of rotation profile as a result of theapplied centrifugal field. With modern instrumentation, theseobservations are electronically digitized and stored for furthermathematical analysis. Two kinds of experiments are commonly performedon these instruments: sedimentation velocity experiments andsedimentation equilibrium experiments.

Sedimentation velocity experiments aim to interpret the entiretime-course of sedimentation, and report on the shape and molar mass ofthe dissolved macromolecules, as well as their size-distribution(Perez-Ramirez and Steckert (2005) Therapeutic Proteins: Methods andProtocols. C. M. Smales and D. C. James, Eds. Vol. 308: 301-318. HumanaPress Inc, Totowa, N.J., US.). The size resolution of this method scalesapproximately with the square of the particle radii, and by adjustingthe rotor speed of the experiment size-ranges from 100 Da to 10 GDa canbe covered. Sedimentation velocity experiments can also be used to studyreversible chemical equilibria between macromolecular species, by eithermonitoring the number and molar mass of macromolecular complexes, bygaining information about the complex composition from multi-signalanalysis exploiting differences in each components spectroscopic signal,or by following the composition dependence of the sedimentation rates ofthe macromolecular system, as described in Gilbert-Jenkins theory.

The kinds of information that can be obtained from an analyticalultracentrifuge include the gross shape of macromolecules, theconformational changes in macromolecules, and size distributions ofmacromolecular samples. For macromolecules, such as proteins, that existin chemical equilibrium with different non-covalent complexes, thenumber and subunit stoichiometry of the complexes and equilibriumconstant constants can be studied. (see also Scott D. J., Harding S. E.and Rowe A. J. Analytical Ultracentrifugation Techniques and Methods,RSC Publishing)

Dynamic light scattering (also known as Photon Correlation Spectroscopyor Quasi-Elastic Light Scattering) is a technique in physics, which canbe used to determine the size distribution profile of small particles insolution. When a beam of light passes through a colloidal dispersion,the particles or droplets scatter some of the light in all directions.When the particles are very small compared with the wavelength of thelight, the intensity of the scattered light is uniform in all directions(Rayleigh scattering); for larger particles (above approximately 250 nmdiameter), the intensity is angle dependent (Mie scattering). If thelight is coherent and monochromatic, as from a laser for example, it ispossible to observe time-dependent fluctuations in the scatteredintensity using a suitable detector such as a photomultiplier capable ofoperating in photon counting mode.

Aggregation can also be measured by the PAMAS SVSS-C(Small VolumeSyringe System-C) instrument (PArtikeIMess—und AnalyseSysteme GMBH),which is a particle size distribution analyzer for low viscous fluids.It uses the principle of light obscuration to detect sub-visibleparticles in the size range 1 μm-200 μm. The validationcriteria/specified limits of the European Pharmacopoeia (EP<2.9.19Particulate Contamination: sub-visible particles) for small and largevolume parenterals are defined by the total counts per container:

The tendency for aggregate formation of a polypeptide in a certainformulation can also be measured by elastic light scattering. Elasticlight scattering can be measured in a spectrofluorometer (e.g.excitation and emission wavelength 500 nm) by temperature-induceddenaturation as measured e.g. at an angle of 90°. Preferably the maximumscatter will stay within the absorption detection limit. The scattershould be 1000 abs. or lower, preferably 750 abs or lower, such as 500abs or lower.

Apart from this and/or in addition, the antigens selected forformulation of the immunogenic composition and the immunogeniccomposition itself may show only low to undetectable levels offragmentation and/or precipitation (insolubility) even during storage.Fragmentation and degradation can be measured e.g. by SE-HPLC and/orRP-HPLC. For example, at least 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%,3%, 4%, 5%, 10%, 20% or 30% of the antigen variant or immunogeniccomposition are degraded, fragmented or insoluble after storage underone or more of the above stress conditions. In some cases at most 0.01%,0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20% or 30% of theantigen variant or immunogenic composition are degraded, fragmented orinsoluble after storage.

Preferably, the antigen variants present in the formulations of thepresent disclosure have a solubility of at least 0.7 mM, at least 0.8mM, at least 0.9 mM, at least 1.0 mM, at least 1.1 mM, at least 1.2 mM,at least 1.3 mM, at least 1.4 mM, at least 1.5 mM, at least 1.6 mM, atleast 1.7 mM, at least 1.8 mM, at least 1.9 mM, at least 2.0 mM, atleast 2.1 mM, at least 2.2 mM, at least 2.3 mM, at least 2.4 mM, atleast 2.5 mM, at least 2.6 mM, at least 2.7 mM, at least 2.8 mM, atleast 2.9 mM, at least 3.0 mM, at least 3.2 mM, at least 3.4 mM, atleast 3.6 mM and/or at least 30 mg/ml, at least 40 mg/ml, at least 50mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml, at least80 mg/ml, at least 90 mg/ml, at least 100 mg/ml, at least 110 mg/ml, atleast 120 mg/ml, at least 130 mg/ml, at least 140 mg/ml, at least 150mg/ml.

The techniques of static light scattering (SLS), tangential flowfiltration (TFF), Fourier Transform Infrared Spectroscopy (FTIR),circular dichroism (CD), urea-induced protein unfolding techniques,intrinsic tryptophan fluorescence, differential scanning calorimetry(DSC), and/or 1-anilino-8-naphthalenesulfonic acid (ANS) protein bindingcan also be used to assess the physical properties and stability ofpolypeptides.

Apart from this and/or in addition, the formulations of the presentinvention show very little to no loss of potency and/or biologicalactivity of their polypeptides, even during storage under one or more ofthe above stress conditions.

Apart from this and/or in addition, the antigens selected forformulation of the immunogenic composition and the immunogeniccomposition itself may show only very little to no loss of potencyand/or biological activity of the antigen (if measurable) even duringstorage. For example, at least 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%,3%, 4%, 5%, 10%, 20% or 30% of the antigen variant or immunogeniccomposition are biologically inactive after storage under one or more ofthe above stress conditions. In some cases at most 0.01%, 0.05%, 0.1%,0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20% or 30% of the antigen variantor immunogenic composition are biologically inactive after storage.

The potency and/or biological activity of a biological describes thespecific ability or capacity of said biological to achieve a definedbiological effect. The potency and biological activities of thepolypeptides of the disclosure can be assessed by various assaysincluding any suitable in vitro assay, cell-based assay, in vivo assayand/or animal model known per se, or any combination thereof, dependingon the specific disease or disorder involved. Suitable in vitro assayswill be clear to the skilled person, and for example include ELISA; FACSbinding assay; Biacore; competition binding assay (AlphaScreen®, PerkinElmer, Massachusetts, USA; FMAT); TRAP assay (osteoclast differentiationassay; Rissanen et al. 2005, J. Bone Miner. Res. 20, Suppl. 1: S256);NF-kappaB reporter gene assay (Mizukami et al. 2002, Mol. Cell. Biol.22: 992-1000).

For example, in one embodiment, Biacore kinetic analysis uses SurfacePlasmon Resonance (SPR) technology to monitor macromolecularinteractions in real time and is used to determine the binding on andoff rates of polypeptides of the formulations of the invention to theirtarget. BIAcore kinetic analysis comprises analyzing the binding anddissociation of the target from chips with immobilized polypeptides ofthe invention on their surface.

E. Immunogenic Composition Formulation

After expression, optional purification and validation of antigenmembers, an ensemble is constructed per the parameters provided for bythe design process. Antigen members of an ensemble are assigned aconcentration in the ensemble such that the concentration of anindividual antigen member is incapable of eliciting an immune response,yet the ensemble as a mixture may be capable of an immune response. Insome cases, an immunogenic composition may comprise an ensemble ofantigen members each provided at the indicated concentration calculatedin the design process. In some cases, the ensemble may be constructedfrom individual antigen members or variants individually expressed,purified and combined at the specified concentration. In other cases,the ensemble may exist as a product of a high throughput expressionmethod such as a phage library, ribosome library or hybrid system. Insome cases, one or more phage libraries, ribosome libraries, in vitrotranslation products, or purified proteins may be combined into anensemble to formulate an immunogenic composition.

i. Pharmaceutical Compositions

In addition to the ensemble of antigen variants, an immunogeniccomposition of the disclosure may comprise any suitable additionalcomponents in generation of a pharmaceutical composition. Apharmaceutically acceptable composition, when administered to a subject,can elicit an immune response against a cell that recognizes the targetepitope across all antigen variants in the ensemble. Thepharmaceutically acceptable compositions of the present disclosure canbe useful as vaccine compositions for prophylactic or therapeutictreatment of a any disease or symptoms thereof.

In some cases, the pharmaceutically acceptable composition furthercomprises a physiologically acceptable carrier, diluent, or excipient.Techniques for formulating and administering also can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,latest edition.

Pharmaceutically acceptable carriers known in the art include, but arenot limited to, sterile water, saline, glucose, dextrose, or bufferedsolutions. Agents such as diluents, stabilizers (e.g., sugars and aminoacids), preservatives, wetting agents, emulsifying agents, pH bufferingagents, additives that enhance viscosity, and the like. Preferably, themedium or carrier will produce minimal or no adverse effects.

In some cases, the pharmaceutically acceptable composition furthercomprises a physiologically acceptable adjuvant. Preferably, theadjuvant employed provides for increased immunogenicity. The adjuvantcan be one that provides for slow release of antigen (e.g., the adjuvantcan be a liposome), or it can be an adjuvant that is immunogenic in itsown right thereby functioning synergistically with antigens. Forexample, the adjuvant can be a known adjuvant or other substance thatpromotes nucleic acid uptake, recruits immune system cells to the siteof administration, or facilitates the immune activation of respondinglymphoid cells. Adjuvants include, but are not limited to,immunomodulatory molecules (e.g., cytokines), oil and water emulsions,aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodiumalginate, Bacto-Adjuvant, synthetic polymers such as poly amino acidsand co-polymers of amino acids, saponin, paraffin oil, and muramyldipeptide.

In some cases, the adjuvant is an immunomodulatory molecule. Forexample, the immunomodulatory molecule can be a recombinant proteincytokine, chemokine, or immunostimulatory agent or nucleic acid encodingcytokines, chemokines, or immunostimulatory agents designed to enhancethe immunologic response.

Examples of immunomodulatory cytokines include interferons (e.g., IFNα,IFNβ and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12 and IL-20), tumor necrosis factors (e.g.,TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1,MIF, MIP-1α, MIP-1β, Rantes, macrophage colony stimulating factor(M-CSF), granulocyte colony stimulating factor (G-CSF), andgranulocyte-macrophage colony stimulating factor (GM-CSF), as well asfunctional fragments of any of the foregoing. Any immunomodulatorychemokine that binds to a chemokine receptor, i.e., a CXC, CC, C, orCX3C chemokine receptor, also can be used in the context of the presentdisclosure. Examples of chemokines include, but are not limited to,Mip1α, Mip-1β, Mip-3α (Larc), Mip-3β, Rantes, Hcc-1, Mpif-1, Mpif-2,Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, Eotaxin, Tarc, Elc, I309, IL-8, Gcp-2Gro-α, Gro-β, Gro-γ, Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, andBca-1 (Blc), as well as functional fragments of any of the foregoing.

In some cases, the adjuvant is a cytokine selected from the groupconsisting of: GM-CSF, G-CSF, IL-2, IL-4, IL-7, IL-12, IL-15, IL-21,TNF-α, and M-CSF. In some cases, the adjuvant is comprised of incompleteFreund's adjuvant (Montanide ISA 51) or Corynebacterium granulosum P40.

In some cases, these adjuvants cannot be expressed from a vector, inwhich case the adjuvant, when used, can be administered simultaneouslyor sequentially, in any order.

In some cases, methods and compositions of the disclosure can be used aspart of combination therapies, for example as methods and/orcompositions comprising one or more other agents such as, but notlimited to, chemotherapeutic, immunotherapeutic, immunomodulatory,anti-angiogenic, anti-viral agents, and hormonal agents.

Examples of anti-viral agents include, but are not limited, aganciclovir (e.g., CYTOVENE®), a valganciclovir (e.g., Valcyte®), afoscarnet (e.g., FOSCAVIR®), a cidofovir (e.g., VISTIDE®, HPMPC), anadefovir (e.g., PMEA, PREVEON®, HEPSERA®), an acyclovir (e.g.,ZOVIRAX®), a valacyclovir (e.g., VALTREX™, ZELITREX™), a polyanion, anda protein kinase C inhibitor (e.g., a bis-indolylmaleide). In one case,the anti-viral agent employed in combination with the compositions andmethods of the present disclosure is a ganciclover, a valganciclovir, acidofovir, or a foscarnet.

In some cases, the one or more other additional agents or components ofthe immunogenic composition can be a chemotherapeutic agent, naturallyoccurring or synthetic, for example as described in “CancerChemotherapeutic Agents”, American Chemical Society, 1995, W. O. FoyeEd. This may be particularly useful if the immunogenic composition isdirected towards cancer.

In some cases, the chemotherapeutic agent is selected from the groupconsisting of a small molecule receptor antagonists such as vatalanib,SU 11248 or AZD-6474, EGFR or HER2 antagonists such as gefitinib,erlotinib, CI-1033 or Herceptin, antibodies such as bevacizumab,cetuximab, rituximab, DNA alkylating drugs such as cisplatin,oxaliplatin or carboplatin, anthracyclines such as doxorubicin orepirubicin, an antimetabolite such as 5-FU, pemetrexed, gemcitabine orcapecitabine, a camptothecin such as irinotecan or topotecan, ananti-cancer drug such as paclitaxel or docetaxel, an epipodophyllotoxinsuch as etoposide or teniposide, a proteasome inhibitor such asbortezomib or anti-inflammatory drugs such as celecoxib or rofecoxib,optionally in form of the pharmaceutically acceptable salts, in form ofthe hydrates and/or solvates and optionally in the form of theindividual optical isomers, mixtures of the individual enantiomers orracemates thereof.

In some cases, chemotherapeutic agent may include but is not limited toa small molecule VEGF receptor antagonist such as vatalanib(PTK-787/ZK222584), SU-5416, SU-6668, SU-11248, SU-14813, AZD-6474,AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 or GW-786034, a dualEGFR/HER2 antagonist such as gefitinib, erlotinib, CI-1033 or GW-2016,an EGFR antagonist such as iressa (ZD-1839), tarceva (OSI-774), PKI-166,EKB-569, HKI-272 or herceptin, an antagonist of the mitogen-activatedprotein kinase such as BAY-43-9006 or BAY-57-9006, a quinazolinederivative such as4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-bute-n-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)quinazolineor4-[(3-chloro-4-fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-bu-ten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline,or a pharmaceutically acceptable salt thereof, a protein kinase receptorantagonist which is not classified under the synthetic small moleculessuch as atrasentan, rituximab, cetuximab, Avastin™ (bevacizumab),IMC-1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin, imatinib, aprotein tyrosine kinase inhibitor which is a fusion protein such asVEGFtrap, an alkylating agent or a platinum compound such as melphalan,cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin,oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin,streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide,streptozocin, thiotepa, chlorambucil, a nitrogen mustard such asmechlorethamine, an ethyleneimine compound, an alkylsulphonate,daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin (doxil),epirubicin, idarubicin, mitoxantrone, amsacrine, dactinomycin,distamycin or a derivative thereof, netropsin, pibenzimol, mitomycin,CC-1065, a duocarmycin, mithramycin, chromomycin, olivomycin, aphtalanilide such as propamidine or stilbamidine, an anthramycin, anaziridine, a nitrosourea or a derivative thereof, a pyrimidine or purineanalogue or antagonist or an inhibitor of the nucleoside diphosphatereductase such as cytarabine, 5-fluorouracile (5-FU), pemetrexed,tegafur/uracil, uracil mustard, fludarabine, gemcitabine, capecitabine,mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin,hydroxyurea, or folic acid, a phleomycin, a bleomycin or a derivative orsalt thereof, CHPP, BZPP, MTPP, BAPP, liblomycin, an acridine or aderivative thereof, a rifamycin, an actinomycin, adramycin, acamptothecin such as irinotecan (camptosar) or topotecan, an amsacrineor analogue thereof, a tricyclic carboxamide, an histonedeacetylaseinhibitor such as SAHA, MD-275, trichostatin A, CBHA, LAQ824, orvalproic acid, an anti-cancer drug from plants such as paclitaxel(taxol), docetaxel or taxotere, a vinca alkaloid such as navelbine,vinblastin, vincristin, vindesine or vinorelbine, a tropolone alkaloidsuch as colchicine or a derivative thereof, a macrolide such asmaytansine, an ansamitocin or rhizoxin, an antimitotic peptide such asphomopsin or dolastatin, an epipodophyllotoxin or a derivative ofpodophyllotoxin such as etoposide or teniposide, a steganacin, anantimitotic carbamate derivative such as combretastatin or amphetinile,procarbazine, a proteasome inhibitor such as bortezomib, an enzyme suchas asparaginase, pegylated asparaginase (pegaspargase) or athymidine-phosphorylase inhibitor, a gestagen or an estrogen such asestramustine (T-66) or megestrol, an anti-androgen such as flutamide,casodex, anandron or cyproterone acetate, an aromatase inhibitor such asaminogluthetimide, anastrozole, formestan or letrozole, a GNrH analoguesuch as leuprorelin, buserelin, goserelin or triptorelin, ananti-estrogen such as tamoxifen or its citrate salt, droloxifene,trioxifene, raloxifene or zindoxifene, a derivative of 17β-estradiolsuch as ICI 164,384 or ICI 182,780, aminoglutethimide, formestane,fadrozole, finasteride, ketoconazole, a LH-RH antagonist such asleuprolide, a steroid such as prednisone, prednisolone,methylprednisolone, dexamethasone, budenoside, fluocortolone ortriamcinolone, an interferon such as interferon β, an interleukin suchas IL-10 or IL-12, an anti-TNFα antibody such as etanercept, animmunomodulatory drug such as thalidomide, its R- and S-enantiomers andits derivatives, or revimid (CC-5013), a leukotrien antagonist,mitomycin C, an aziridoquinone such as BMY-42355, AZQ or EO-9, a2-nitroimidazole such as misonidazole, NLP-1 or NLA-1, a nitroacridine,a nitroquinoline, a nitropyrazoloacridine, a “dual-function” nitroaromatic such as RSU-1069 or RB-6145, CB-1954, a N-oxide of nitrogenmustard such as nitromin, a metal complex of a nitrogen mustard, ananti-CD3 or anti-CD25 antibody.

In some cases, the chemotherapeutic agent is selected from the groupconsisting of compounds interacting with or binding tubulin, syntheticsmall molecule VEGF receptor antagonists, small molecule growth factorreceptor antagonists, inhibitors of the EGF receptor and/or VEGFreceptor and/or integrin receptors or any other protein tyrosine kinasereceptors which are not classified under the synthetic small-molecules,inhibitors directed to EGF receptor and/or VEGF receptor and/or integrinreceptors or any other protein tyrosine kinase receptors, which arefusion proteins, compounds which interact with nucleic acids and whichare classified as alkylating agents or platinum compounds, compoundswhich interact with nucleic acids and which are classified asanthracyclines, as DNA intercalators or as DNA cross-linking agents,including DNA minor-groove binding compounds, anti-metabolites,naturally occurring, semi-synthetic or synthetic bleomycin typeantibiotics, inhibitors of DNA transcribing enzymes, and especially thetopoisomerase I or topoisomerase II inhibitors, chromatin modifyingagents, mitosis inhibitors, anti-mitotic agents, cell-cycle inhibitors,proteasome inhibitors, enzymes, hormones, hormone antagonists, hormoneinhibitors, inhibitors of steroid biosynthesis, steroids, cytokines,hypoxia-selective cytotoxins, inhibitors of cytokines, lymphokines,antibodies directed against cytokines, oral and parenteral toleranceinduction agents, supportive agents, chemical radiation sensitizers andprotectors, photo-chemically activated drugs, synthetic poly- oroligonucleotides, optionally modified or conjugated, non-steroidalanti-inflammatory drugs, cytotoxic antibiotics, antibodies targeting thesurface molecules of cancer cells, antibodies targeting growth factorsor their receptors, inhibitors of metalloproteinases, metals, inhibitorsof oncogenes, inhibitors of gene transcription or of RNA translation orprotein expression, complexes of rare earth elements, andphoto-chemotherapeutic agents.

In some cases, the chemotherapeutic agent is selected from the groupconsisting of paclitaxel (taxol), docetaxel, a vinca alkaloid such asnavelbine, vinblastin, vincristin, vindesine or vinorelbine, analkylating agent or a platinum compound such as melphalan,cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin,oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin,streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide,streptozocin, thiotepa, chlorambucil, a nitrogen mustard such asmechlorethamine, an immunomodulatory drug such as thalidomide, its R-and S-enantiomers and its derivatives, or revimid (CC-5013)), anethyleneimine compound, an alkylsulphonate, daunorubicin, doxorubicin(adriamycin), liposomal doxfflubicin (doxil), epirubicin, idarubicin,mitoxantrone, amsacrine, dactinomycin, distamycin or a derivativethereof, netropsin, pibenzimol, mitomycin, CC-1065, a duocarmycin,mithramycin, chromomycin, olivomycin, a phtalanilide such as propamidineor stilbamidine, an anthramycin, an aziridine, a nitrosourea or aderivative thereof, a pyrimidine or purine analogue or antagonist or aninhibitor of the nucleoside diphosphate reductase such as cytarabine,5-fluorouracile (5-FU), uracil mustard, fludarabine, gemcitabine,capecitabine, mercaptopurine, cladribine, thioguanine, methotrexate,pentostatin, hydroxyurea, or folic acid, an acridine or a derivativethereof, a rifamycin, an actinomycin, adramycin, a camptothecin such asirinotecan (camptosar) or topotecan, an amsacrine or analogue thereof, atricyclic carboxamide, an histonedeacetylase inhibitor such as SAHA,MD-275, trichostatin A, CBHA, LAQ824, or valproic acid, or a proteasomeinhibitor such as bortezomib.

In some cases, the chemotherapeutic agent is a compound which reducesthe transport of hyaluronan mediated by one or more ABC transporters, ordrug transport inhibitor, such as a P-glycoprotein (P-gp) inhibitormolecule or inhibitor peptide, an MRP1 inhibitor, an antibody directedagainst and capable of blocking the ABC transporter, an antisenseoligomer, iRNA, siRNA or aptamer directed against one or more ABCtransporters. Examples of P-glycoprotein (P-gp) inhibitor molecules inaccordance with the present disclosure are zosuquidar (LY 335973), itssalts (especially the trichloride salt) and its polymorphs, cyclosporinA (also known as cyclosporine), verapamil or its R-isomer, tamoxifen,quinidine, d-alpha tocopheryl polyethylene glycol 1000 succinate,VX-710, PSC833, phenothiazine, GF120918 (II), SDZ PSC 833, TMBY, MS-073,S-9788, SDZ 280-446, XR(9051) and functional derivatives, analogues andisomers of these.

ii. Haptens and Scaffolds

Ensembles comprising a plurality of antigen variants may also compriseadditional components. In some instances, antigen variants may befurther derivatized to include a hapten. Haptens, or small, lowmolecular weight molecules may be attached to antigens, or in particularone or more epitopes on a particular antigen variant. As known in theart, haptens may be any low molecular weight molecule or moiety. In somecases, a hapten may comprise a phosphate group linked an amino acid.This may include but is not limited to phosphothreonine orphosphotyrosine. In some cases, artificial amino acids phosphothreonineand phosphotyrosine or mimics of phosphothreonine and phosphotyrosinemay be incorporated directly into the antigen variant. IN some casesthese haptens may be in or around the target epitope.

In other cases, haptens may be drugs or small molecules which may becrosslinked or affinity bound to the antigen variant, or target epitopeon the antigen variant. In some cases, haptens may include but are notlimited to drugs such as hallucinogens, for example mescaline and LSD;cannabinoids, for example THC; dissociative drugs such asPCP/phencyclidine and ketamine; stimulants, for example amphetamines,cocaine, phenmetrazine, methylphenidate; nicotine; depressants, forexample, nonbarbiturates (e.g. bromides, chloral hydrate etc.),methaqualone, barbiturates, diazepam, flurazepam, phencyclidine, andfluoxetine; opium and its derivatives, for example, heroin, methadone,morphine, meperidine, codeine, pentazocine, and propoxyphene;prescription drugs including opioids (for pain), central nervous systemdepressants (for anxiety and sleep disorders), and stimulants (for ADHDand narcolepsy). Opioids include hydrocodone (Vicodin®), oxycodone(OxyContin®), propoxyphene

(Darvon®), hydromorphone (Dilaudid®), meperidine (Demerol®), anddiphenoxylate (Lomotil®). Central nervous system depressants includebarbiturates such as pentobarbital sodium (Nembutal®), andbenzodiazepines such as diazepam, (Valium®) and alprazolam (Xanax®).Stimulants include dextroamphetamine (Dexedrine®), methylphenidate(Ritalin® and Concerta®), and amphetamines (Adderall®); club drugsinclude GHB, Rohypnol®, ketamine, and others; and “designer drugs” suchas “ecstasy.”

Further, antigen variants, may be bound or fused to additional moleculesor proteins known as scaffolds. Generally scaffolds include moleculeswhich themselves are not immunogenic, but provide suitable platform onwhich to express or attach various antigen variants. For example, phagecoat proteins may be considered scaffold proteins. In some cases,antigen variants may be irreversibly or reversibly linked to phage coatproteins. In other cases, scaffolds may other proteins such as otherviral coat proteins, protein complexes capable of oligomerization. Insome cases, scaffolds may be other organic or inorganic molecules whichmay polymerize, such as dendrimers. In some cases, scaffolds may beuseful for immunogenic composition such as vaccines, whereby theimmunogen (i.e immugenic composition comprising the ensemble) isconcentrated in a single molecule, rather than dispersed.

iii. DNA Based Formulations

In some cases, the immunogenic composition of the disclosure maycomprise nucleic acids encoding the antigen variant proteins of theimmunogenic composition. In some cases, nucleic acids may beadministered to a subject or immune cell rather than purified proteins.In some cases, a subject or immune cell generates the antigen variantprotein from nucleic acids delivered to the subject or immune cell

The compositions and methods of the disclosure provide for any suitabledelivery vector of antigen variant encoding nucleic acids to cells in ahuman subject or patient in need thereof.

In some cases, delivery of the nucleic acid may be performed using anysuitable “vector” (sometimes also referred to as “gene delivery” or“gene transfer vehicle). Vector, delivery vehicle, gene delivery vehicleor gene transfer vehicle, may refer to any suitable macromolecule orcomplex of molecules comprising a polynucleotide to be delivered to atarget cell. In some cases, a target cell may be any cell to which thenucleic acid or gene is delivered.

For example, suitable vectors may include but are not limited to, viralvectors such as adenoviruses, adeno-associated viruses (AAV), andretroviruses, liposomes, other lipid-containing complexes, and othermacromolecular complexes capable of mediating delivery of apolynucleotide to a target cell.

In some cases, a vector may be an organic or inorganic molecule. In somecases, a vector may be small molecule (i.e. <5 kD), or a macromolecule(i.e. >5 kD). For example a vector may include but is not limited toinert, non-biologically active molecules such as metal particles. Insome cases, a vector may be gold particles.

In some cases a vector may comprise a biologically active molecule. Forexample, vectors may comprise polymerized macromolecules such asdendrimers.

In some cases, a vector may comprise a recombinant viral vector thatincorporates one or more nucleic acids. As described herein, nucleicacids may refer to polynucleotides. Nucleic acid and polynucleotide maybe used interchangeably. In some cases nucleic acids may comprise DNA orRNA. In some cases RNA nucleic acids may include but are not limited toa transcript of a gene of interest (e.g. antigen variant sequence),intronsuntranslated regions, termination sequences and the like. Inother cases, DNA nucleic acids may include but are not limited tosequences such as hybrid promoter gene sequences, strong constitutivepromoter sequences, the antigen variant of interest, untranslatedregions, termination sequences and the like. In some cases, acombination of DNA and RNA may be used.

As described in the disclosure herein, the term “expression construct”is meant to include any type of genetic construct containing a nucleicacid or polynucleotide coding for gene products in which part or all ofthe nucleic acid encoding sequence is capable of being transcribed. Thetranscript may be translated into a protein. In some cases it may bepartially translated or not translated. In certain aspects, expressionincludes both transcription of a gene and translation of mRNA into agene product. In other aspects, expression may only includetranscription of the nucleic acid encoding antigen variants of theimmunogenic composition.

In some cases, a plurality of nucleic acids may be delivered by suitablevectors, each nucleic acid encoding one or more antigen members orvariants of the immunogenic composition. Individual concentrations ofantigen member proteins may be controlled by suitable expression controlelements, such as promoters, enhancers, repressors and the like. In someinstances, the administration of nucleic acids to generate antigenvariant proteins of an immunogenic composition in a subject or immunecell may be refered to as a “nucleic acid vaccine.”

iv. General Formulation Methods

Generally, the immunogenic compositions of this disclosure may bepurified from culture supernatant using a process wherein the clarifiedsupernatant (obtained by centrifugation) is captured on any combinationof columns selected from (without being limiting) affinitychromatography resin such as Protein A resin, Cation ExchangeChromatography (CIEC) or an Anion Exchange Chromatography (AIEC) usingfor example Poros SOHS (FORDS), SOURCE 30S or SOURCE 15S (GEHealthcare), SP Sepharose (GE Healthcare), Capto S (GE Healthcare),Capto MMC (GE Healthcare) or Poros 50HQ (POROS), SOURCE 30Q or SOURCE15Q (GE Healthcare), Q Sepharose (GE Healthcare), Capto Q and DEAESepharose (GE Healthcare), Size exclusion chromatography (SE-HPLC) usingfor example Superdex 75 or Superdex 200 (GE Healthcare), hydrophobicinteraction chromatography (HIC) using for example octyl, butylsepharose or equivalents, optionally also including a tangential flowfiltration (TFF) step. Any combination of columns can be used for thepurification of the polypeptides of the invention, such as e.g. ProteinA resin followed by Cation Exchange Chromatography or two CationExchange Chromatography steps.

The present invention also provides methods for preparing the stableformulations of the invention comprising the ensembles of the invention.More particularly, the present invention provides methods for preparingstable formulations of such ensembles, said methods comprisingconcentrating a fraction containing the purified antigen variants to afinal concentration of more than 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml,70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, or 150 mg/ml, such as e.g. 65mg/ml using a semipermeable membrane with an appropriate molecularweight (MW) cutoff and diafiltering and/or ultrafiltering to bufferexchange and further concentrate the polypeptide fraction into theformulation buffer using the same membrane.

The pH of the formulation may range from 3.0 to about 12.0. The pH ofthe immunogenic composition may be at least 3, 4, 5, 6, 7, 8, 9, 10, 11or 12 pH units. The pH of the immunogenic composition may be at most 3,4, 5, 6, 7, 8, 9, 10, 11 or 12 pH units.

The formulations of the present invention may be sterilized by varioussterilization methods, including sterile filtration, radiation, etc. Ina specific embodiment, the polypeptide formulation is filter-sterilizedwith a presterilized 0.2 micron filter.

The formulations of the invention may be lyophilized (freeze dried) ifdesired. Accordingly, the invention also encompasses lyophilized formsof the formulations of the invention. Preferably the final residualwater content of the lyophilized formulation is extremely low, around 1%to 4%.

The formulations of the invention may also be spray dried if desired.Accordingly, the invention also encompasses spray dried forms of theformulations of the invention. Preferably the final residual watercontent of the spray dried formulation is extremely low, around 1% to4%.

Spray drying is a method of producing a dry powder from a liquid orslurry by rapidly drying with a hot gas. Spray drying works bydispersing the liquid formulation into a controlled drop size spray andpassing hot air as the heated drying media.

In some cases, the liquid, lyophilized or spray dried formulation of thepresent invention is supplied in a hermetically sealed container. Liquidformulations may comprise a quantity between 1 ml and 20 ml. In somecases, liquid formulations may be at least 1 ml, 2 ml, 3 ml, 4 ml, 5 ml,6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml.

The liquid, lyophilized or spray dried formulations of the presentinvention can be prepared as unit dosage forms by preparing a vialcontaining an aliquot of the liquid, lyophilized or spray driedformulation for a one time use. For example, a unit dosage of liquidformulation per vial may contain 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml of the formulation. In apreferred aspect, the unit dosage form is suitable for subcutaneousadministration to a subject. In another aspect, the subject is a human.

The amount of a formulation of the present invention which will beeffective in the prevention, treatment and/or management of a certaindisease or disorder can be determined by standard clinical techniqueswell-known in the art or described herein, The precise dose to beemployed in the formulation will also depend on the route ofadministration, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. For formulations of the polypeptide, encompassed bythe invention, the dosage administered to a patient may further becalculated using the patient's weight in kilograms (kg) multiplied bythe dose to be administered in mg/kg.

The required volume (in ml) to be given is then determined by taking themg dose required divided by the concentration of the polypeptideformulation. The final calculated required volume will be obtained bypooling the contents of as many vials as are necessary into syringe(s)to administer the polypeptide formulation of the invention.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture or kit includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. In oneembodiment, the unit dosage form is suitable for intravenous,intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus,the invention encompasses formulations, preferably sterile, suitable foreach delivery route. In the case of dosage forms suitable for parenteraladministration (such as e.g. subcutaneous administration) the activeingredient, e.g., polypeptide of the invention is sterile and suitablefor administration as a particulate free solution. In other words, theinvention encompasses both parenteral solutions and lyophilized or spraydried powders, each being sterile, and the latter being suitable forreconstitution prior to injection.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises the formulation containing the polypeptide. Thepackaging material includes instruction means which indicate that saidpolypeptide can be used to prevent, treat and/or manage one or moresymptoms associated with the disease or disorder by administeringspecific doses and using specific dosing regimens as described herein.

The invention also provides an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of each pharmaceutical agent containedwithin said packaging material, wherein one pharmaceutical agentcomprises a formulation containing the polypeptide of interest, andwherein said packaging material includes instruction means whichindicate that said agents can be used to prevent, treat and/or managethe disease or disorder by administering specific doses and usingspecific dosing regimens as described herein.

The formulations, containers, pharmaceutical unit dosages and kits ofthe present invention may be administered to a subject to prevent, treatand/or manage a specific disease and/or disorder in subject in needthereof.

V. Administration of the Immunogenic Composition

After an immunogenic composition is formulated from an ensemble of aplurality of antigen variants and other optional components as describedherein, the immunogenic composition may be administered to a subject,animal or cell to induce an immune response.

The pharmaceutical unit dosage forms can be made suitable for any formof delivery of the polypeptide of the invention including (without beinglimiting) parenteral delivery, topical delivery, pulmonary delivery,intranasal delivery, vaginal delivery, enteral delivery, rectaldelivery, oral delivery and/or sublingual delivery. In one aspect, thepresent invention relates to a pharmaceutical unit dosage form suitablefor parenteral (such as e.g. intravenous, intraarterial, intramuscular,intracerebral, intraosseous, intradermal, intrathecal, intraperitoneal,subcutaneous, etc) administration to a subject, comprising a formulationof the invention in a suitable container. In some cases, an immunogeniccomposition may be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 20 times to a subject. In some cases, an immunogenic composition maybe administered at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 times to asubject.

Generally, any animal that possesses an immune system may be used toadminister an immunogenic composition. For example an immunogeniccomposition of the disclosure may be administered to a human, anon-human primate, livestock, a mammal, a bovine, equine, porcine,ovine, caprine, feline, canine, buffalo, guinea pig, hamster, rabbit,mice, fish, shark, bird or reptile subject.

Administering an immunogenic composition of this disclosure to an animalor subject may be performed for a variety of reasons. In some cases,immunogenic compositions may be administered as a vaccine or fortherapeutic applications as described herein.

In some cases, the immunogenic composition may also used to harvestantibodies from a host animal. In some cases, animals just as goats,mice, rodents, chickens and rabbits may be used specifically forantibody harvest and purification as described in the art. In somecases, one or more of these animals may be administered the immunogeniccomposition. In some cases, the host immune response generatescirculating antibodies in the serum of these animals. Harvesting theserum of these animals may be useful in generating and isolating anantibody of interest.

In some cases, an immunogenic composition may be administered or used toexpose an immune cell. In some cases, cultured immune cells, such as Tcell, B cell, dendritic cells, antigen presenting cells, or any suitableimmune cell, may be cultured and exposed to immunogenic composition invitro. In this case, the immune cell may elicit an immune response tothe immunogenic composition in vitro. In some cases, immune cells maycomprise a cultured cell line. In some cases immune cells may beextracted and harvested from a patient. As described herein, the methodsand compositions of the disclosure may be used to “train” immune cellsof a subject, such as used in a therapeutic application. In some cases,immune cells may be isolated and extracted from a subject, contactedwith an immunogenic composition as provided by the disclosure, andreturned to the subject.

In another example, immune cells may be used to test the immunogenicresponse of the immunogenic composition. As described herein, techniquessuch as FACS may be used to test the specificity and/or immunogenicityof a plurality of antigen variants when contacted with immune cells suchas B cells.

In another example, such as with a hybridoma, or immortalized B cell,this cell results from a fusion between a B cell and an immortalizedcell line such as cancer cell line, and may be used. An immunogeniccomposition may be applied to a culture of hybridomas. In some cases,recognition of the target epitope by one or more hybridoma cells mayelicit an immune response in the form on antibody production. In somecases, antibodies produced in this way may be purified and used.Generally, any immune cell may be used in this manner. In someinstances, exposure to an immune cell may be used to test an immunogeniccomposition, or in some cases it may be used to elicit a response forisolation of a particular antibody to target epitope.

IV. Applications A. Vaccine Development

i. Vaccine Strategies

The composition and methods of this disclosure provide for numerousapplications. One important application is the development of improvedvaccines towards any pathogenic, cancer, autoimmune antigens or smallmolecules, such as drugs.

In some cases, an antigen may be derived (e.g., selected) from apathogenic organism. In some cases, the antigen is a cancer or tumorantigen, e.g., an antigen derived from a tumor or cancer cell.

In some cases, an antigen derived from a pathogenic organism is anantigen associated with an infectious disease; it can be derived fromany of a variety of infectious agents, including virus, bacterium,fungus or parasites.

In some cases, a target antigen is any antigen associated with apathology, for example an infectious disease or pathogen, or cancer oran immune disease, inflammatory disease, addiction disease, neurologicaldisease, or autoimmune disease. In some cases, an antigen can beexpressed by any of a variety of infectious agents, including virus,bacterium, fungus or parasite. A target antigen for use in the methodsand compositions as disclosed herein can also include, for example,pathogenic peptides, toxins, toxoids, subunits thereof, or combinationsthereof (e.g., cholera toxin, tetanus toxoid).

The present disclosure provides for a plurality of antigen variantsselected from a computation-guided library. In some cases, vaccines ofthe disclosure may also comprise recombinant proteins or peptides,carbohydrates, glycoproteins, glycopeptides, proteoglycans, inactivatedorganisms, and viruses, dead organisms and virus, genetically alteredorganisms or viruses, or cell extracts. In some aspects, an immunogeniccomposition may comprise nucleic acids, carbohydrates, lipids, and/orsmall molecules. In some aspects, an immunogenic composition is one thatelicits an immune response. In other aspects, an immunogenic compositionis a polynucleotide that encodes a protein or peptide that when theprotein or peptide is expressed an immune response is elicited. In someaspects, an immunogenic composition is an antigen. In some aspects, animmunogenic composition is a protein or peptide. In some aspects, animmunogenic composition is used for vaccines.

Any of the antigens described herein may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, glycopeptides,proteoglycans, nucleic acids that encode a protein or peptide,carbohydrates, small molecules, or combinations thereof. In someaspects, an immunogenic composition is derived from a microorganism forwhich at least one vaccine already exists. In some aspects, animmunogenic composition is derived from a microorganism for which novaccines have been developed.

In some aspects, an immunogenic composition or vaccine of the disclosurecomprises two types of anitgens which are both derived from a singlegenus of microorganism. In some aspects, an immunogenic composition orvaccine of the disclosure comprises two types of immunogeniccompositions which are both derived from a single genus and species ofmicroorganism. In some aspects, an immunogenic composition or vaccine ofthe disclosure comprises two types of antigens which are both derivedfrom a single genus, species, and strain of microorganism. In someaspects, a immunogenic composition or vaccine of the disclosurecomprises two types of antigens which are both derived from a singleclone of a microorganism.

In some aspects, an immunogenic composition or vaccine of the disclosurecomprises two or more types of antigens which are derived from differentstrains of a single species of microorganism. In some aspects, animmunogenic composition or vaccine of the disclosure comprises two ormore types of antigens which are derived from different species of thesame genus of microorganism. In other aspects, an immunogeniccomposition or vaccine of the disclosure comprises two or more types ofantigens each derived from different genera of microorganism.

In some aspects, a immunogenic composition or vaccine of the disclosurecomprises a single type of antigen that elicits an immune response inboth B cells and T cells. In some aspects, an immunogenic composition orvaccine of the disclosure comprises two types of antigens, wherein thefirst immunogenic composition stimulates B cells, and the second type ofantigen stimulates T cells. In some aspects, one or both anitgens maystimulate T cells and B cells. In some aspects, an immunogeniccomposition or vaccine of the disclosure comprises greater than twotypes of antigens, wherein one or more types of antigens stimulate Bcells, and one or more types of antigens stimulate T cells.

In some aspects, the immunogenic composition comprises a T cell antigenor plurality of antigen variants, and the T cell antigen is derived fromthe same pathogen against which vaccination is intended. In this case,an initially small number of naive T cells are stimulated to generatepathogen-specific effector and memory T cells. In some aspects, theantigen may be taken from an unrelated source, such as an infectiousagent to which wide-spread immunity already exists (e.g., tetanus toxoidor a common component of influenza virus, such as hemagglutinin,neuraminidase, or nuclear protein). In the latter case, the vaccineexploits the presence of memory T cells that have arisen in response toprior infections or vaccinations. Memory cells in general react morerapidly and vigorously to antigen rechallenge and, therefore, mayprovide a superior source of help to B cells.

Other antigens include, but are not limited to, degenerative diseaseantigens, infectious disease antigens, cancer antigens, allergens,alloantigens, atopic disease antigens, autoimmune disease antigens,contact sensitizers, haptens, xenoantigens, or metabolic disease enzymesor enzymatic products thereof and as described herein.

ii. Antigen Selection

In some cases, a vaccine may be developed using the composition andmethods of the disclosure, to antigens selected from Picornaviridae (forexample, polio viruses, hepatitis A virus; enteroviruses, humancoxsackie viruses, rhinoviruses, echoviruses); Calciviridae (such asstrains that cause gastroenteritis); Togaviridae (for example, equineencephalitis viruses, rubella viruses); Flaviridae (for example, dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae (forexample, coronaviruses); Rhabdoviridae (for example, vesicularstomatitis viruses, rabies viruses); Filoviridae (for example, ebolaviruses); Paramyxoviridae (for example, parainfluenza viruses, mumpsvirus, measles virus, respiratory syncytial virus); Orthomyxoviridae(for example, influenza viruses); Bungaviridae (for example, Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviursesand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpessimplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus(CMV), Marek's disease virus, herpes viruses); Poxviridae (variolaviruses, vaccinia viruses, pox viruses); and Iridoviridae (such asAfrican swine fever virus); and unclassified viruses (for example, theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk andrelated viruses, and astroviruses). The compositions and methodsdescribed herein are contemplated for use in treating infections withthese viral agents.

In some cases, a vaccine may be developed using the composition andmethods of the disclosure, to antigens selected from fungal pathogenswhich include but are not limited to aspergillosis; thrush (caused byCandida albicans); cryptococcosis (caused by Cryptococcus); andhistoplasmosis. Thus, examples of infectious fungi include, but are notlimited to, Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis,Candida albicans. Components of these organisms can be included asantigens in the MAPS described herein.

In some cases, a vaccine may be developed, using the compositions andmethods of the disclosure, to antigens selected from infectious microbesincluding but not limited to Bordatella pertussis, Brucella, Enterococcisp., Neisseria meningitidis, Neisseria gonorrheae, Moraxella, typeableor nontypeable Haemophilus, Pseudomonas, Salmonella, Shigella,Enterobacter, Citrobacter, Klebsiella, E. coli, Helicobacter pylori,Clostridia, Bacteroides, Chlamydiaceae, Vibrio cholera, Mycoplasma,Treponemes, Borelia burgdorferi, Legionella pneumophilia, Mycobacteriasps (such as M. tuberculosis, M. avium, M. intracellular e, M. kansaii,M. gordonae, M. leprae), Staphylococcus aureus, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus anthracis, Corynebacteriumdiphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Leptospira sps., Pasteurella multocida,Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis,Treponema pallidium, Treponema pertenue, and Actinomyces israelii. Thecompositions and methods described herein are contemplated for use intreating or preventing infections against these bacterial agents.

Additional parasite pathogens from which antigens can be derivedinclude, for example: Entamoeba histolytica, Plasmodium falciparum,Leishmania sp., Toxoplasma gondii, Rickettsia, and the Helminths.

In some cases, a vaccine may be developed, using the compositions andmethods of the disclosure, to antigens selected from a truncatedpneumococcal PsaA protein, pneumolysin toxoid pneumococcalserine/threonine protein kinase (StkP), pneumococcal serine/threonineprotein kinase repeating unit (StkPR), pneumococcal PcsB protein,staphylococcal alpha hemolysin, Mycobacterium tuberculosis mtb proteinESAT-6, M. tuberculosis cell wall core antigen, Chlamydia CT144, CT242or CT812 polypeptides or fragments of these, Chlamydia DNA gyrasesubunit B, Chlamydia sulfite synthesis/biphosphate phosphatase,Chlamydia cell division protein FtsY, Chlamydia methionyl-tRNAsynthetase, Chlamydia DNA helicase (uvrD), Chlamydia ATP synthasesubunit I (atpl), or Chlamydia metal dependent hydrolase.

In some cases, a vaccine may be developed, using the compositions andmethods of the disclosure, to antigens selected from the pathogenMyocobacterium tuberculosis (TB), an intracellular bacterial parasite.One example of a TB antigen is TbH9 (also known as Mtb 39A). Other TBantigens include, but are not limited to, DPV (also known as Mtb8.4),381, Mtb41, Mtb40, Mtb32A, Mtb64, Mtb83, MA9.9A, Mtb9.8, Mtb16, Mtb72f,Mtb59f, Mtb88f, Mtb71f, Mtb46f and Mtb31f, wherein “f’ indicates that itis a fusion or two or more proteins.

In some cases, a vaccine may be developed, using the compositions andmethods of the disclosure, to antigens selected from Chlamydia speciesfor use in the immunogenic compositions of the present disclosure.Chlamydiaceae (consisting of Chlamydiae and Chlamydophila), are obligateintracellular gram-negative bacteria. Chlamydia trachomatis infectionsare among the most prevalent bacterial sexually transmitted infections,and perhaps 89 million new cases of genital chlamydial infection occureach year. The Chlamydia of the present disclosure include, for example,C. trachomatis, Chlamydophila pneumoniae, C muridarum, C. suis,Chlamydophila abortus, Chlamydophila psittaci,

Chlamydophila caviae, Chlamydophila felis, Chlamydophila pecorum, and Cpneumoniae. Animal models of chlamydial infection have established thatT-cells play a critical role both in the clearance of the initialinfection and in protection from re-infection of susceptible hosts.Hence, the immunogenic compositions as disclosed herein can be used toprovide particular value by eliciting cellular immune responses againstchlamydial infection.

More specifically, Chlamydial antigens useful in the present disclosureinclude DNA gyrase subunit B, sulfite synthesis/biphosphate phosphatase,cell division protein FtsY, methionyl-tRNA synthetase, DNA helicase(uvrD); ATP synthase subunit I (atpl) or a metal-dependent hydrolase(U.S. Patent Application Pub. No. 20090028891). Additional Chlamydiatrachomatis antigens include CT144 polypeptide, a peptide having aminoacid residues 67-86 of CT144, a peptide having amino acid residues 77-96of CT144, CT242 protein, a peptide having amino acids 109-117 of CT242,a peptide having a mino acids 112-120 of CT242 polypeptide, CT812protein (from the pmpD gene), a peptide having amino acid residues103-111 of the CT812 protein; and several other antigenic peptides fromC trachomatis:

In some cases, a vaccine may be developed, using the compositions andmethods of the disclosure, to antigens selected from fungal antigensderived from Candida species and other yeast; or other fungi(aspergillus, other environmental fungi). Regarding other parasites,malaria as well as worms and amoebae may provide the antigenic antigenfor use in the in the immunogenic compositions and methods as disclosedherein.

In some cases, an antigen for use in the immunogenic compositions asdisclosed herein can also include those used in biological warfare, suchas ricin, which may provoke an immune response.

Additionally, the present disclosure also provides immunogeniccompositions comprising antigens which raise an immune response againstcancer. In these conjugates, an antigen is an antigen expressed by acancer or tumor, or derived from a tumor. In some cases, such antigensare referred to herein as a “cancer antigen” and are typically a proteinexpressed predominantly on the cancer cells, such that the conjugateelicits both potent humoral and potent cellular immunity to thisprotein. A large number of cancer-associated antigens have beenidentified, several of which are now being used to make experimentalcancer treatment vaccines and are thus suitable for use in the presentcases. Antigens associated with more than one type of cancer includeCarcinoembryonic antigen (CEA); Cancer/testis antigens, such asNY-ESO-1; Mucin-1 (MUC1) such as Sialyl Tn (STn); Gangliosides, such asGM3 and GD2; p53 protein; and HER2/neu protein (also known as ERBB2).Antigens unique to a specific type of cancer include a mutant form ofthe epidermal growth factor receptor, called EGFRvIII;Melanocyte/melanoma differentiation antigens, such as tyrosinase, MARTI,gplOO, the lineage related cancer-testis group (MAGE) andtyrosinase-related antigens; Prostate-specific antigen;Leukaemia-associated antigens (LAAs), such as the fusion proteinBCR-ABL, Wilms' tumour protein and proteinase 3; and Idiotype (Id)antibodies. See, e.g., Mitchell, 3 Curr. Opin. Investig. Drugs 150(2002); Dao & Scheinberg, 21 Best Pract. Res. Clin.

Haematol. 391 (2008).

Another approach in generating an immune response against cancer employsantigens from microbes that cause or contribute to the development ofcancer. These vaccines have been used against cancers includinghepatocellular carcinoma (hepatitis B virus, hepatitis C virus,Opisthorchis viverrin), lymphoma and nasoparyngeal carcinoma(Epstei-Barr virus), colorectal cancer, stomach cancer (Helicobacterpylori), bladder cancer (Schisosoma hematobium), T-cell leukemia (humanT-cell lymphtropic virus), cervical cancer (human papillomavirus), andothers. To date, there have been clinical trials for vaccines targetingBladder Cancer, Brain Tumors, Breast Cancer, Cervical Cancer, KidneyCancer, Melanoma, Multiple Myeloma, Leukemia, Lung Cancer, PancreaticCancer, Prostate Cancer, and Solid Tumors. See Pardoll et al., ABELOFF'SCLIN. ONCOL. (4th ed., Churchill Livingstone, Philadelphia 2008); Sioud,360 Methods Mol. Bio. 277 (2007); Pazdur et al., 30 J. Infusion Nursing30(3): 173 (2007); Parmiani et al., 178 J. Immunol. 1975 (2007); Lolliniet al., 24 Trends Immunol. 62 (2003); Schlom et al., 13 Clin. CancerRes. 3776 (2007); Banchereau et al., 392 Nature 245 (1998); Finn, 358New Engl. J. Med. 2704 (2008); Curigliano et al., 7 Exp. Rev. AnticancerTher. 1225 (2007). Marek's Disease virus, a herpes virus that causestumors in poultry, has long been managed by vaccine. Thus, the presentcases encompass both preventive or prophylactic anti-cancer immunogeniccompositions and treatment/therapeutic cancer vaccines.

In some cases, a vaccine may be developed, using the compositions andmethods of the disclosure, to antigens associated with proliferativediseases and cancers which include AIDS related cancers, acousticneuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocysticcarcinoma, adrenocortical cancer, agnogenic myeloid metaplasia,alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma,astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladdercancer, bone cancers, bowel cancer, brain and CNS tumors, breast cancer,carcinoid tumors, cervical cancer, childhood brain tumours, childhoodcancer, childhood leukemia, childhood soft tissue sarcoma,chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronicmyeloid leukemia, colorectal cancers, cutaneous t-cell lymphoma,dermatofibrosarcoma-protuberans, desmoplastic-small-round-cell-tumour,ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma,esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eyecancer, including, e.g., eye melanoma and retinoblastoma, fallopian tubecancer, fanconi anemia, fibrosarcoma, gall bladder cancer, gastriccancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumour,genitourinary cancers, germ cell tumors, gestational-trophoblasticdisease, glioma, gynecological cancers, hematological malignancies,hairy cell leukemia, head and neck cancer, hepatocellular cancer,hereditary breast cancer, Hodgkin's disease, humanpapillomavirus-related cervical cancer, hydatidiform mole, hypopharynxcancer, islet cell cancer, Kaposi's sarcoma, kidney cancer, laryngealcancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer,liposarcoma, lung cancer, lymphedema, lymphoma, non-Hodgkin's lymphoma,male breast cancer, malignant-rhabdoid-tumour-of-kidney,medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastaticcancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides,myelodysplastic syndromes, myeloma, myeloproliferative disorders, nasalcancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma,neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin cancer,non-small-cell-lung-cancer-(NSCLC), oral cavity cancer, oropharynxcancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasalcancer, parathyroid cancer, parotid gland cancer, penile cancer,peripheral-neuroectodermal-tumours, pituitary cancer, polycythemia vera,prostate cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma,Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, Schwannoma,Sezary syndrome, skin cancer, small cell lung cancer (SCLC), smallintestine cancer, soft tissue sarcoma, spinal cord tumours,squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,testicular cancer, thymus cancer, thyroid cancer,transitional-cell-cancer-(bladder), transitional-cell-cancer(renal-pelvis/ureter), trophoblastic cancer, urethral cancer, urinarysystem cancer, uterine sarcoma, uterus cancer, Vaginal cancer, vulvacancer, Waldenstrom's-macroglobulinemia, and Wilms' tumor.

In some cases, a vaccine may be developed, using the compositions andmethods of the disclosure, to antigens associated with autoimmunediseases, e.g., they can be “self-antigens.” Autoimmune diseasescontemplated for diagnosis according to the assays described hereininclude, but are not limited to alopecia areata, ankylosing spondylitis,antiphospholipid syndrome, Addison's disease, aplastic anemia, multiplesclerosis, autoimmune disease of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune oophoritis and orchitis,Behcet's Disease, bullous pemphigoid, cardiomyopathy, celiacsprue-dermatitis, chronic fatigue syndrome, chronic inflammatorydemyelinating syndrome (CFIDS), chronic inflammatory polyneuropathy,Churg-Strauss syndrome, cicatricial pemphigoid, CREST Syndrome, coldagglutinin disease, Crohn's disease, dermatitis herpetiformis, discoidlupus, essential mixed cryoglobulinemia, fibromyalgia,glomerulonephritis, Grave's disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA nephropathy, insulin dependent diabetes (Type I),Lichen Planus, lupus, Meniere's Disease, mixed connective tissuedisease, myasthenia gravis, myocarditis, pemphigus vulgaris, perniciousanemia, polyarteritis nodosa, polychondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud'sphenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome,Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, Wegener's syndrome, vasculitis and vitiligo. It isgenerally important to assess the potential or actual CMI responsivenessin subjects having, or suspected of having or being susceptible to anautoimmune disease.

In some cases, an antigen for use in the immunogenic compositions asdisclosed herein can be an antigen which is associated with aninflammatory disease or condition. Examples of inflammatory diseaseconditions where antigens may be useful include but are not limited toacne, angina, arthritis, aspiration pneumonia, empyema, gastroenteritis,necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis,pleurisy, chronic inflammatory demyelinating polyneuropathy, chronicinflammatory demyelinating polyradiculoneuropathy, and chronicinflammatory demyelinating polyneuropathy.

In some cases, an antigen for use in the immunogenic compositions asdisclosed herein can be an antigen which is associated with aninflammatory disease or condition.

The compositions and methods of the present disclosure provide forvaccinations that may be either passive or active in nature. In general,active vaccinations may involve the exposure of a subject's immunesystem to one or more agents that are recognized as unwanted, undesired,and/or foreign and elicit an endogenous immune response resulting in theactivation of antigen-specific naive lymphocytes that then give rise toantibody-secreting B cells or antigen-specific effector and memory Tcells or both. This approach can result in long-lived protectiveimmunity that may be boosted from time to time by renewed exposure tothe same antigenic material, such as an immunogenic composition of thedisclosure. In some cases, the compositions and methods of thisdisclosure may provide for a recipient or subject to be injected withpreformed antibodies or with antigen-specific effector lymphocytes,which may confer rapid ad hoc protection, but generally do not establishpersistent immunity.

Some current vaccines against, e.g., microbial pathogens, consist oflive attenuated or non-virulent variant strains of microorganisms, orkilled or otherwise inactivated organisms. Other vaccines utilize moreor less purified components of pathogen lysates, such as surfacecarbohydrates or recombinant pathogen-derived proteins that aresometimes fused to other molecules, particularly proteins that canconfer adjuvant activity. In some cases, one or more of the vaccinepreparations may be used with the compositions and methods of thedisclosure.

iii. Vaccination Process

Vaccines that utilize live attenuated or inactivated pathogens can, insome cases, yield a vigorous immune response, but their use may havelimitations. For example, live vaccine strains can sometimes causeinfectious pathologies, especially when administered toimmune-compromised recipients. Moreover, many pathogens, particularlyviruses, undergo continuous rapid mutations in their genome, which allowthem to escape immune responses to antigenically distinct vaccinestrains. However, most or all pathogens are thought to possess someantigenic determinants that are not easily mutated because they areassociated with essential functions. Antibodies directed against theseconserved epitopes, rather than more variable, non-essential epitopescan protect against highly mutable viruses (Baba et al, 2000, Nat. Med.,6:200; incorporated herein by reference) may be suitable targetepitopes/target antigens as provided by the compositions and methods ofthe disclosure. Vaccines based on live or killed intact pathogens do notnecessarily promote the recognition of these critical epitopes, but mayessentially “distract” the immune system to focus its assault on highlyvariable determinants. In some cases the present disclosure provides forengineered vaccines that help an immune response focus on a particularimmunogenic part of some antigens, and may present selectivelyessential, immutable or substantially immutable epitopes. In some cases,this may provide more potent and “escape-proof neutralizing antibody andeffector T cell responses than intact microorganisms.

The precise mechanisms by which vaccines stimulate antibody responses indraining lymph nodes (or fail to do so) are complex. B and T cells maybe initially sequestered in distinct anatomic regions, the superficiallylocated B follicles and the surrounding paracortex and deep cortex,respectively in some mammalian systems. Upon antigen challenge,antigen-specific B cells in follicles as well as CD4 T cells in the Tcell area may become activated and then migrate toward the border zonebetween the two compartments. B cells that have phagocytosed lymph-borneantigens process the acquired material and begin to present antigenicpeptides in MHC class-II surface molecules that are then recognized bythe activated CD4 T cells (the TF_(H) cells). Antigen-recognition allowsthe TF_(H) cells to provide help to B cells, which constitutes a potentsurvival signal and triggers the formation of germinal centers (GCs)within B follicles. The GC reaction promotes class-switch recombination,affinity maturation of antigen-specific antibodies, and the formation ofmemory B cells and long-lived plasma cells that can produce largeamounts of high-affinity antibodies for extended periods of time. Thus,the present disclosure provides for an immunogenic composition orvaccine that may comprise components that allow antigenic material to beefficiently recognized by both B and T cells and in some cases to inducevigorous GC reactions.

Immunogenic compositions may be exposed to distinct cells of the immunesystem and stimulate them. In some aspects, immunogenic compositions ofthe present disclosure stimulate B cells, and immunogenic compositionsmay be processes by antigen-presenting cells (APCs), such as dendriticcells (DCs), in lymphoid tissues (and by B cells after activation) andpresented to T cells.

In some aspects, immunogenic compositions may be modified such that thesurface of one or more antigen variants may be attached or associatedwith a targeting moiety (e.g., antibody or fragment thereof, peptide orpolypeptide, Affibody, Nanobody™, AdNectin™, Avimer™, aptamer,Spiegelmer, small molecule, lipid, carbohydrate, etc.). In some casesimmunogenic compositions of the disclosure may be targeted to specificantigen presenting cells, such as DCs, SCS-Mph, FDCs, T Cells, B cells,and/or combinations thereof.

iv. Targeting Moieties for T Cells

In some aspects, vaccines of the present disclosure comprise at leastone immunogenic composition which can be delivered to APCs, which thenprocess and deliver the immunogenic composition(s) to T cells.Professional APCs are very efficient at internalizing antigen, either byphagocytosis or by endocytosis, and then display a fragment of theantigen, bound to either a class II major histocompatibility complex(class II MHC) molecule or a class I MHC molecule on the APC membrane.CD4 T cells recognize and interact with the antigen-class II MHCmolecule complex on the APC membrane, whereas CD8 T cells recognize andinteract with the antigen-class I MHC molecule complex. An additionalco-stimulatory signal as well as modulating cytokines are then producedby the APC, leading to T cell activation.

In some aspects, immunogenic compositions comprise one or more targetingmoieties. A targeting moiety is any moiety that binds to a componentassociated with an organ, tissue, cell, extracellular matrix, and/orsubcellular locale. In some aspects, such a component is referred to asa “target” or a “marker,” and these are discussed in further detailbelow.

A targeting moiety may be a nucleic acid, polypeptide, glycoprotein,carbohydrate, lipid, small molecule, etc. For example, a targetingmoiety can be a nucleic acid targeting moiety (e.g. an aptamer,Spiegelmer®, etc.) that binds to a cell type specific marker. Ingeneral, an aptamer is an oligonucleotide (e.g., DNA, RNA, or an analogor derivative thereof) that binds to a particular target, such as apolypeptide. In some aspects, a targeting moiety may be a naturallyoccurring or synthetic ligand for a cell surface receptor, e.g., agrowth factor, hormone, LDL, transferrin, etc. A targeting moiety can bean antibody, which term is intended to include antibody fragments,characteristic portions of antibodies, single chain antibodies, etc.Synthetic binding proteins such as Affibodies®, Nanobodies™, AdNectins™,Avimers™, etc., can be used. Peptide targeting moieties can beidentified, e.g., using procedures such as phage display. This widelyused technique has been used to identify cell specific ligands for avariety of different cell types.

In accordance with the present disclosure, a targeting moiety recognizesone or more “targets” or “markers” associated with a particular organ,tissue, cell, and/or subcellular locale. In some aspects, a target maybe a marker that is exclusively or primarily associated with one or afew cell types, with one or a few diseases, and/or with one or a fewdevelopmental stages.

In some aspects, a target is a tumor marker. In some aspects, a tumormarker is an antigen that is expressed in tumor cells but not in healthyand/or normal cells. In some aspects, a tumor marker is an antigen thatis more prevalent in tumor cells than in healthy and/or normal cells.Exemplary tumor markers include, but are not limited to, gplOO; Melan-A;tyrosinase; PSMA; HER-2/neu; MUC-I; topoisomerase Ilα; sialyl-Tn;carcinoembryonic antigen; ErbB-3-binding protein-1; alpha-fetoprotein;and the cancer-testis antigens MAGE-Al, MAGE A4, and NY-ESO-I.

In some aspects, a target is an APC marker. In some aspects, a target isa DC marker. In some aspects, a target is a T cell marker. In someaspects, a T cell target is an antigen that is expressed in T cells butnot in non-T cells. In some aspects, a T cell target is an antigen thatis more prevalent in T cells than in non-T cells.

In some aspects, targeting moieties are covalently associated with oneor more antigen variants of an immunogenic composition of thedisclosure. In some aspects, covalent association is mediated by alinker. In some aspects, targeting moieties are not covalentlyassociated with an antigen variant. For example, targeting moieties maybe associated with the surface of, encapsulated within, surrounded by,and/or distributed throughout the polymeric matrix of an inventiveparticle.

Dendritic Cells (DCs) are a type of myeloid leukocytes; they are amongthe most potent antigen presenting cells for T lymphocytes. Resting DCsreside in many tissues, including lymph nodes, in an immature,tolerogenic state, i.e., they present intermediate to high levels ofpeptide-MHC complexes, but with little or no costimulatory molecules andwithout secreting cytokines that T cells need to differentiate intoeffector cells. T cells that are presented with a specific antigen byimmature DCs begin to proliferate for a few days, but then they die byapoptosis or become unresponsive to further activation. The ensuingdepletion of antigen-specific T cell responses renders the hostselectively tolerant to this antigen. By contrast, when DCs acquireantigens while they are exposed to maturation stimuli, the cells rapidlyup-regulate MHC and costimulatory molecules and secrete severalcytokines. The now mature DCs are potent inducers of effector T cellsand immunological memory.

DC targeting can be accomplished by moieties including but not limitedto molecules that bind DC-205, CDl Ic, class II MHC, CD80, CD86,DC-SIGN, CDl Ib, BDCA-I, BDCA-2, BDCA-4, Siglec-H, CX3CR1, and/orLangerin.

In some aspects, DC targeting can be accomplished by any targetingmoiety that specifically binds to any entity (e.g., protein, lipid,carbohydrate, small molecule, etc.) that is prominently expressed and/orpresent on DCs (i.e., a DC marker). Exemplary DC markers include, butare not limited to, CDIa (R4, T6, HTA-I); CDIb (RI); CDIc (M241, R7);CDId (R3); CDIe (R2); CDl Ib (aM Integrin chain, CR3, MoI, C3niR,Mac-1); CDl Ic (aX Integrin, p150, 95, AXb2); CDwI 17 (Lactosylceramide,LacCer); CD19 (B4); CD33 (gp67); CD 35 (CR1, C3b/C4b receptor); CD 36(GpIIIb, GPIV, PASIV); CD39 (ATPdehydrogenase, NTPdehydrogenase-1); CD40(Bp50); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO(UCHL-I); CD49d (VLA-4α, α4 Integrin); CD169 (Sialoadhesin, Siglec-1);CD208 (DC-LAMP); CD209 (DC-SIGN); CDw218a (IL18Rα); CDw218b (IL8Rβ);CD227 (MUCl, PUM, PEM, EMA); CD230 (Prion Protein (PrP)); CD252 (OX40L,TNF (ligand) superfamily, member 4); CD258 (LIGHT, TNF (ligand)superfamily, member 14); CD265 (TRANCE-R, TNF-R superfamily, member 1Ia); CD271 (NGFR, p75, TNFR superfamily, CD283 (TLR3, TOLL-like receptor3); CD300c (CMRF-35A); CD301 (MGL1, CLECSF14); CD302 (DCL1); CD303(BDCA2); CD304 (BDCA4); CD312 (EMR2); CD317 (BST2); CD319 (CRACC,SLAMF7); CD320 (8D6); and CD68 (gpl 10, Macrosialin); class II MHC.

In some aspects, T cell targeting can be accomplished by any targetingmoiety that specifically binds to any entity (e.g., protein, lipid,carbohydrate, small molecule, etc.) that is prominently expressed and/orpresent on T cells (i.e., a T cell marker). Exemplary T cell markersinclude, but are not limited to, CD2 (E-rosette R, Tl 1, LFA-2); CD3(T3); CD3 α; CD3 13; CD3 ε; CD4 (L3T4, W3/25, T4); CD5 (T1, Tp67, Leu-1,LY-I); CD6 (T 12); CD7 (gp40, Leu 9); CD8a (Leu2, T8, Lyt2,3); CD8b(CD8, Leu2, Lyt3); CDl Ia (LFA-lα, α Integrin chain); CDl Ib (αMIntegrin chain, CR3, MoI, C3niR, Mac-1); CDl Ic (αX Integrin, p150, 95,AXb2); CD15s (Sialyl Lewis X); CD15u (3′ sulpho Lewis X); CD15su (6sulpho-sialyl Lewis X); CD 16b (FcgRlllb); CDw 17 (Lactosylceramide,LacCer); CD 18 (Integrin β2 CDl Ia, b, c β-subunit); CD26 (DPP IVectoeneyme, ADA binding protein); CD27 (T14, S 152); CD28 (Tp44, T44);CD29 (Platelet GPlIa, β-1 integrin, GP); CD31 (PECAM-I, Endocam); CD35(CRl, C3b/C4b receptor); CD37 (g_(p)52-40); CD38 (ADP-ribosyl/cyclase,T10); CD43 (Sialophorin, Leukosialin).

v. Immunostimulatory Agents

In some aspects, immunogenic composition or vaccine of the disclosuresare formulated with one or more adjuvants such as gel-type adjuvants(e.g., aluminum hydroxide, aluminum phosphate, calcium phosphate, etc.),microbial adjuvants (e.g., immunomodulatory DNA sequences that includeCpG motifs; endotoxins such as monophosphoryl lipid A; exotoxins such ascholera toxin, E. coli heat labile toxin, and pertussis toxin; muramyldipeptide, etc.); oil-emulsion and emulsifier-based adjuvants (e.g.,Freund's Adjuvant, MF59 [Novartis], SAF, etc.); particulate adjuvants(e.g., liposomes, biodegradable microspheres, saponins, etc.); syntheticadjuvants (e.g., nonionic block copolymers, muramyl peptide analogues,polyphosphazene, synthetic polynucleotides, etc.), and/or combinationsthereof. Other exemplary adjuvants include some polymers (e.g.,polyphosphazenes, described in U.S. Pat. No. 5,500,161, which isincorporated herein by reference), QS21, squalene, tetrachlorodecaoxide,etc.

vi. Assays for T Cell Activation

In some aspects, various assays can be utilized in order to determinewhether an immune response has been elicited in a T cell or group of Tcells (i.e., whether a T cell or group of T cells has become“activated”). In some aspects, stimulation of an immune response in Tcells can be determined by measuring antigen-induced production ofcytokines by T cells. In some aspects, stimulation of an immune responsein T cells can be determined by measuring antigen-induced production ofIFNγ, IL-4, IL-2, IL-IO, IL-17 and/or TNFα by T cells. In some aspects,antigen-produced production of cytokines by T cells can be measured byintracellular cytokine staining followed by flow cytometry. In someaspects, antigen-induced production of cytokines by T cells can bemeasured by surface capture staining followed by flow cytometry. In someaspects, antigen-induced production of cytokines by T cells can bemeasured by determining cytokine concentration in supernatants ofactivated T cell cultures. In some aspects, this can be measured byELISA.

In some aspects, antigen-produced production of cytokines by T cells canbe measured by ELISPOT assay. In general, ELISPOT assays employ atechnique very similar to the sandwich enzyme-linked immunosorbent assay(ELISA) technique. An antibody {e.g. monoclonal antibody, polyclonalantibody, etc.) is coated aseptically onto a PVDF (polyvinylidenefluoride)-backed microplate. Antibodies are chosen for their specificityfor the cytokine in question. The plate is blocked {e.g. with a serumprotein that is non-reactive with any of the antibodies in the assay).Cells of interest are plated out at varying densities, along withantigen or mitogen, and then placed in a humidified 37° C. CO₂ incubatorfor a specified period of time. Cytokine secreted by activated cells iscaptured locally by the coated antibody on the high surface area PVDFmembrane. After washing the wells to remove cells, debris, and mediacomponents, a secondary antibody {e.g., a biotinylated polyclonalantibody) specific for the cytokine is added to the wells. This antibodyis reactive with a distinct epitope of the target cytokine and thus isemployed to detect the captured cytokine. Following a wash to remove anyunbound biotinylated antibody, the detected cytokine is then visualizedusing an avidin-HRP, and a precipitating substrate {e.g., AEC,BCIP/NBT). The colored end product (a spot, usually a blackish blue)generally represents an individual cytokine-producing cell. Spots can becounted manually {e.g., with a dissecting microscope) or using anautomated reader to capture the microwell images and to analyze spotnumber and size. In some aspects, each spot correlates to a singlecytokine-producing cell.

In some aspects, an immune response in T cells is said to be stimulatedif between at least 1% and at least 100% of antigen-specific T cellsproduce cytokines. In some aspects, an immune response in T cells issaid to be stimulated if at least 1%, at least 5%, at least 10%, atleast 25%, at least 50%, at least 75%, at least 90%, at least 95%, atleast 99%, or at least 100% of antigen-specific T cells producecytokines.

In some aspects, an immune response in T cells is said to be stimulatedif immunized subjects comprise at least 10-fold, at least 50-fold, atleast 100-fold, at least 500-fold, at least 1000-fold, at least5000-fold, at least 10,000-fold, at least 50,000-fold, at least100,000-fold, or greater than at least 100,000-fold morecytokine-producing cells than do naive controls.

In some aspects, stimulation of an immune response in T cells can bedetermined by measuring antigen-induced proliferation of T cells. Insome aspects, antigen-induced proliferation may be measured as uptake ofH³-thymidine in dividing T cells (sometimes referred to as “lymphocytetransformation test, or “LTT”). In some aspects, antigen-inducedproliferation is said to have occurred if H³-thymidine uptake (given asnumber of counts from a γ counter) is at least 5-fold, at least 10-fold,at least 20-fold, at least 50-fold, at least 100-fold, at least500-fold, at least 1000-fold, at least 5000-fold, at least 10,000-fold,or greater than at least 10,000-fold higher than a naïve control.

In some aspects, antigen-induced proliferation may be measured by flowcytometry. In some aspects, antigen-induced proliferation may bemeasured by a carboxyfluorescein succinimidyl ester (CFSE) dilutionassay. CFSE is a non-toxic, fluorescent, membrane-permeating dye thatbinds the amino groups of cytoplasmic proteins with itssuccinimidyl-reactive group (e.g. T cell proteins). When cells divide,CFSE-labeled proteins are equally distributed between the daughtercells, thus halving cell fluorescence with each division. Consequently,antigen-specific T cells lose their fluorescence after culture in thepresence of the respective antigen (CFSE^(low)) and are distinguishablefrom other cells in culture (CFSE^(high)). In some aspects,antigen-induced proliferation is said to have occurred if CFSE dilution(given as the percentage of CFSE^(low) cells out of all CFSE⁺ cells) isat least 5%, at least 10%, at least 25%, at least 50%, at least 75%, atleast 90%, at least 95%, or at least 100%.

In some aspects, an immune response in T cells is said to be stimulatedif cellular markers of T cell activation are expressed at differentlevels (e.g. higher or lower levels) relative to unstimulated cells. Insome aspects, CDl Ia CD27, CD25, CD40L, CD44, CD45RO, and/or CD69 aremore highly expressed in activated T cells than in unstimulated T cells.In some aspects, L-selectin (CD62L), CD45RA, and/or CCR7 are less highlyexpressed in activated T cells than in unstimulated T cells.

In some aspects, an immune response in T cells is measured by assayingcytotoxicity by effector CD8⁺ T cells against antigen-pulsed targetcells. For example, a ⁵¹chrornium (⁵¹Cr) release assay can be performed.In this assay, effector CD8⁺ T cells bind infected cells presentingvirus peptide on class I MHC and signal the infected cells to undergoapoptosis. If the cells are labeled with ⁵¹Cr before the effector CD8⁺ Tcells are added, the amount Of ⁵¹Cr released into the supernatant isproportional to the number of targets killed.

One of ordinary skill in the art will recognize that the assaysdescribed above are only exemplary methods which could be utilized inorder to determine whether T cell activation has occurred. Any assayknown to one of skill in the art which can be used to determine whetherT cell activation has occurred falls within the scope of thisdisclosure. The assays described herein as well as additional assaysthat could be used to determine whether T cell activation has occurredare described in Current Protocols in Immunology (John Wiley & Sons,Hoboken, N Y, 2007; incorporated herein by reference).

vii. Targeting B Cells

The present disclosure provides immunogenic composition or vaccine ofthe disclosures for delivery of, for example, immunogenic compositionsto the cells of the immune system. In some aspects, immunogeniccomposition or vaccine of the disclosures comprise at least oneimmunogenic composition which can be presented to B cells (i.e., B cellantigens).

As described herein, one or more antigen variants of an immunogeniccomposition of the disclosure may comprise one or more targetingmoieties. In some aspects, targeting moieties target particular celltypes. In some aspects, a target is a B cell marker. In some aspects, aB cell target is an antigen that is expressed in B cells but not innon-B cells. In some aspects, a B cell target is an antigen that is moreprevalent in B cells than in non-B cells.

In some aspects, a target is a SCS-Mph marker. In some aspects, anSCS-Mph target is an antigen that is expressed in SCS-Mph but not innon-SCS-Mph. In some aspects, an SCS-Mph target is an antigen that ismore prevalent in SCS-Mph than in non-SCS-Mph. Exemplary SCS-Mph markersare listed below in the section entitled “Subcapsular Sinus MacrophageCells” and include those provided elsewhere herein. In some aspects, atarget is a FDC marker. In some aspects, an FDC target is an antigenthat is expressed in FDCs but not in non-FDCs. In some aspects, an FDCtarget is an antigen that is more prevalent in FDCs than in non-FDCs.Exemplary FDC markers are listed below in the section entitled“Follicular Dendritic Cells.” and include those provided elsewhereherein.

In some aspects, a target is preferentially expressed in particular celltypes. For example, expression of an SCS-Mph, FDC, and/or B cell targetin SCS-Mph, FDCs, and/or B cells is at least 2-fold, at least 3-fold, atleast 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, atleast 50-fold, at least 100-fold, at least 500-fold, or at least1000-fold overexpressed in SCS-Mph, FDCs, and/or B cells relative to areference population. In some aspects, a reference population maycomprise non-SCS-Mph, FDCs, and/or B cells.

The present disclosure encompasses the recognition that targeting ofantigens to subcapsular sinus macrophages (SCS-Mph) is involved inefficient early presentation of lymph-borne pathogens, such as viruses,to follicular B cells (FIG. 2). As described in Example 1, followingsubcutaneous injection of vesicular stomatitis virus (VSV) or adenovirus(AdV) into the footpad of mice, viral particles were efficiently andselectively retained by CD169⁺ SCS-Mph in the draining popliteal lymphnodes. VSV-specific B cell receptor (BCR) transgenic B cells in theselymph nodes were rapidly activated and generated extremely high antibodytiters upon this viral challenge. Depletion of SCS-Mph by injection ofliposomes laden with clodronate (which is toxic for Mph) abolished earlyB cell activation, indicating that SCS-Mph are essential for thepresentation of lymph-borne particulate antigens to B cells.

In some aspects, SCS-Mph targeting can be accomplished by any targetingmoiety that specifically binds to any entity (e.g., protein, lipid,carbohydrate, small molecule, etc.) that is prominently expressed and/orpresent on macrophages (i.e., SCS-Mph markers). Exemplary SCS-Mphmarkers include, but are not limited to, CD4 (L3T4, W3/25, T4); CD9(p24, DRAP-I, MRP-I); CDl Ia (LFA-1α, α L Integrin chain); CDl Ib (aMIntegrin chain, CR3, MoI, C3niR, Mac-1); CD1 Ic (αX Integrin, p150, 95,AXb2); CDwl 2 (p90-120); CD13 (APN, gp150, EC 3.4.11.2); CD14 (LPS-R);CD15 (X-Hapten, Lewis, X, SSEA-I, 3-FAL); CD15s (Sialyl Lewis X); CD15u(3′ sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD16a (FCRIIIA);CD16b (FcgRIIIb); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin β2,CDl 1a,b,c β-subunit); CD26 (DPP IV ectoeneyme, ADA binding protein);CD29 (Platelet GPIIa, β-1 integrin, GP); CD31 (PECAM-I, Endocam); CD32(FCγRII); CD33 (gp67); CD35 (CRl, C3b/C4b receptor); CD36 (GpIIIb, GPIV,PASIV); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, T10); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD43 (Sialophorin,Leukosialin); CD44 (EMCRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5);CD45RA; CD45RB; CD45RC; CD45RO (UCHL-I); CD46 (MCP); CD47 (gp42, IAP,OA3, Neurophillin); CD47R (MEM-133); CD48 (Blast-1, Hulym3, BCM-1,OX-45); CD49a (VLA-lα, α1 Integrin); CD49b (VLA-2α, gpla, α2 Integrin);or CD49c (VLA-3α, α3 Integrin).

In some aspects, SCS-Mph targeting can be accomplished by any targetingmoiety that specifically binds to any entity (e.g., protein, lipid,carbohydrate, small molecule, etc.) that is prominently expressed and/orpresent on macrophages upon activation (i.e., activated SCS-Mph marker).Exemplary activated SCS-Mph markers include, but are not limited to,CDIa (R4, T6, HTA-I); CDIb (RI); CDIc (M241, R7); CD44R (CD44v, CD44v9);CD49d (VLA-4α, α4 Integrin); CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD105 (Endoglin); CD 142 (Tissue factor, Thromboplastin, F3); CD 143 (ACE,Peptidyl dipeptidase A, Kininase II); CD153 (CD3OL, TNSF8); CD163 (M130,GHI/61, RM3/1); CD 166 (ALCAM, KG-CAM, SC-I, BEN, DM-GRASP); CD227(MUCl, PUM, PEM, EMA); CD253 (TRAIL, TNF (ligand) superfamily, member10); CD273 (B7DC, PDL2); CD274 (B7H1, PDLl); CD275 (B7H2, ICOSL); CD276(B7H3); CD297 (ART4, ADP-ribosyltransferase 4; and Dombrock blood groupglycoprotein; wherein the names listed in parentheses representalternative names. Examples of such markers include those providedelsewhere herein.

In some aspects, B cell targeting can be accomplished by moieties thatbind the complement receptors, CR1 (i.e., CD35) or CR2 (i.e., CD21),proteins which are expressed on B cells as well as FDCs. In someaspects, B cell targeting can be accomplished by B cell markers such asCD 19, CD20, and/or CD22. In some aspects, B cell targeting can beaccomplished by B cell markers such as CD40, CD52, CD80, CXCR5, VLA-4,class II MHC, surface IgM or IgD, APRL, and/or BAFF-R. The presentdisclosure encompasses the recognition that simultaneous targeting of Bcells by moieties specific for complement receptors or otherAPC-associated molecules boosts humoral responses.

B cells that initially detect a previously unknown antigen generallyexpress a B cell receptor (BCR, i.e., an antibody with a transmembranedomain) with suboptimal binding affinity for that antigen. However, Bcells can increase by several orders of magnitude the affinity of theantibodies they make when they enter into a germinal center (GC)reaction. This event, which generally lasts several weeks, depends onFDC that accumulate, retain and present antigenic material to theactivated B cells. B cells, while proliferating vigorously, repeatedlymutate the genomic sequences that encode the antigen binding site oftheir antibody and undergo class-switch recombination to form secretedhigh-affinity antibodies, mostly of the IgG isotype. GC reactions alsostimulate the generation of long-lived memory B cells and plasma cellsthat maintain high protective antibody titers, often for many years.Vaccine that target FDC upon subcutaneous injection and that areretained on the FDC surface for long periods of time are predicted toboost GC reactions in response to vaccination and improve the affinityand longevity of desired humoral immune responses.

In some aspects, FDC targeting can be accomplished by moieties that bindthe complement receptors, CR1 (i.e., CD35) or CR2 (i.e., CD21), proteinswhich are expressed on FDCs as well as B cells. Examples of moietiesinclude those provided elsewhere herein.

GC reactions and B cell survival not only require FDC, but also aredependent on help provided by activated CD4 T cells. Help is mostefficiently provided when a CD4 T cell is first stimulated by a DC thatpresents a cognate peptide in MHC class II (pMHC) to achieve afollicular helper (T_(FH)) phenotype. The newly generated TFH cell thenmigrates toward the B follicle and provides help to those B cells thatpresent them with the same pMHC complex. For this, B cells first acquireantigenic material (e.g., virus or virus-like vaccine), internalize andprocess it (i.e., extract peptide that is loaded into MHC class II), andthen present the pMHC to a TF_(H) cell.

Thus, the present disclosure encompasses the recognition that a vaccinethat elicits optimal humoral immunity can combine several features andcomponents: (a) antigenic material for CD4 T cells that is targeted toand presented by DCs; (b) high density surface antigens that can bepresented in their native form by SCS-Mph to antigen-specific follicularB cells; (c) the capacity to be acquired and processed by follicular Bcells for presentation to T_(FH) cells (the present disclosureencompasses the recognition that B cells readily acquire and internalizeparticulate matter from SCS-Mph); (d) the ability to reach FDC and beretained on FDC in intact form and for long periods of time; and (e)adjuvant activity to render APC fully immunogenic and to avoid orovercome tolerance.

In some aspects, an immunogenic composition or vaccine of the disclosurecomprises at least one targeting moiety. In some aspects, all of thetargeting moieties of an immunogenic composition or vaccine of thedisclosure are identical to one another. In some aspects, an immunogeniccomposition or vaccine of the disclosure a number of different types oftargeting moieties. In some aspects, an immunogenic composition orvaccine of the disclosure comprises multiple individual targetingmoieties, all of which are identical to one another. In some aspects, aimmunogenic composition or vaccine of the disclosure comprises exactlyone type of targeting moiety. In some aspects, a immunogenic compositionor vaccine of the disclosure comprises exactly two distinct types oftargeting moieties. In some aspects, a immunogenic composition orvaccine of the disclosure comprises greater than two distinct types oftargeting moieties.

In some aspects, a immunogenic composition or vaccine of the disclosurecomprises at least one targeting moiety that is associated with theexterior surface of the immunogenic composition or vaccine of thedisclosure. In some aspects, the association is covalent. In someaspects, the covalent association is mediated by one or more linkers. Insome aspects, the association is non-covalent. In some aspects, thenon-covalent association is mediated by charge interactions, affinityinteractions, metal coordination, physical adsorption, host-guestinteractions, hydrophobic interactions, TT stacking interactions,hydrogen bonding interactions, van der Waals interactions, magneticinteractions, electrostatic interactions, dipole-dipole interactions,and/or combinations thereof.

viii. Assays for B Cell Activation

In some aspects, various assays can be utilized in order to determinewhether an immune response has been elicited in a B cell or group of Bcells (i.e., whether a B cell or group of B cells has become“activated”). In some aspects, stimulation of an immune response in Bcells can be determined by measuring antibody titers. In general,“antibody titer” refers to the ability of antibodies to bind andneutralize antigens at particular dilutions. For example, a highantibody titer refers to the ability of antibodies to bind andneutralize antigens even at high dilutions. In some aspects, an immuneresponse in B cells is said to be stimulated if antibody titers aremeasured to be positive at dilutions at least 5-fold greater, at least10-fold greater, at least 20-fold greater, at least 50-fold greater, atleast 100-fold greater, at least 500-fold greater, at least 1000 foldgreater, or more than at least 1000-fold greater than in non-immunizedindividuals or pre-immune serum.

In some aspects, stimulation of an immune response in B cells can bedetermined by measuring antibody affinity. In particular, an immuneresponse in B cells is said to be stimulated or elictied if an antibodyhas an equilibrium dissociation constant (Kd) less than 10⁻⁷ M, lessthan 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻¹⁰ M, less than 10⁻¹¹ M,less than 10¹² M, or less.

In some aspects, a T cell-dependent immune response in B cells is saidto be stimulated if class-switch recombination has occurred. Inparticular, a switch from IgM to an IgG isotype or to IgA or to amixture of these isotypes is indicative of a T cell dependent immuneresponse in B cells.

In some aspects, an immune response in B cells is determined bymeasuring affinity maturation of antigen-specific antibodies. Affinitymaturation occurs during the germinal center reaction whereby activatedB cells repeatedly mutate a region of the immunoglobulin gene thatencodes the antigen-binding region. B cells producing mutated antibodieswhich have a higher affinity for antigen are preferentially allowed tosurvive and proliferate. Thus, over time, the antibodies made by B cellsin GCs acquire incrementally higher affinities. In some aspects, thereadout of this process is the presence of high antibody titer (e.g.high affinity IgG antibodies that bind and neutralize antigens even athigh dilutions).

In some aspects, an immune response in B cells is said to be stimulatedif memory B cells and/or long-lived plasma cells that can produce largeamounts of high-affinity antibodies for extended periods of time haveformed. In some aspects, antibody titers are measured after differenttime intervals (e.g. 2 weeks, 1 month, 2 months, 6 months, 1 year, 2years, 5 years, 10 years, 15 years, 20 years, 25 years, or longer) aftervaccination in order to test for the presence of memory B cells and/orlong-lived plasma cells that can produce large amounts of high-affinityantibodies for extended periods of time. In some aspects, memory B cellsand/or long-lived plasma cells that can produce large amounts ofhigh-affinity antibodies for extended periods of time are said to bepresent by measuring humoral responses (e.g., if humoral responses aremarkedly more rapid and result in higher titers after a later boostervaccination than during the initial sensitization). In some aspects, animmune response in B cells is said to be stimulated if a vigorousgerminal center reaction occurs.

In some aspects, a vigorous germinal center reaction can be assessedvisually by performing histology experiments. In some aspects, vigorousgerminal center reaction can be assayed by performingimmunohistochemistry of antigen-containing lymphoid tissues (e.g.,vaccine-draining lymph nodes, spleen, etc.). In some aspects,immunohistochemistry is followed by flow cytometry.

In some aspects, an immune response in B cells is determined byanalyzing antibody function in neutralization assays. In particular, theability of a microorganism (e.g., virus, bacterium, fungus, protozoan,parasite, etc.) to infect a susceptible cell line in vitro in theabsence of serum is compared to conditions when different dilutions ofimmune and nonimmune serum are added to the culture medium in which thecells are grown. In some aspects, an immune response in a B cell is saidto be stimulated if infection of a microorganism is neutralized at adilution of at least 1:5, at least 1:10, at least 1:50, at least 1:100,at least 1:500, at least 1:1000, at least 1:5000, at least 1:10,000, orless.

In some aspects, the efficacy of vaccines in animal models may bedetermined by infecting groups of immunized and non-immunized mice(e.g., 3 or more weeks after vaccination) with a dose of a microorganismthat is generally lethal. The magnitude and duration of survival of bothgroup is monitored and generally graphed a Kaplan-Meier curves. Toassess whether enhanced survival is due to B cell responses, serum fromimmune mice can be transferred as a “passive vaccine” to assessprotection of nonimmune mice from lethal infection.

One of ordinary skill in the art will recognize that the assaysdescribed above are only exemplary methods which could be utilized inorder to determine whether B cell activation has occurred. Any assayknown to one of skill in the art which can be used to determine whetherB cell activation has occurred falls within the scope of thisdisclosure. The assays described herein as well as additional assaysthat could be used to determine whether B cell activation has occurredare described in Current Protocols in Immunology (John Wiley & Sons,Hoboken, N Y, 2007; incorporated herein by reference).

ix. Determination of Immune Response by Sequencing

In some cases, an elicited immune response to an immunogenic compositionof the disclosure may be performed by direct sequencing of immune cells,including T cella do B cells. For example, to determine if B cells or Tcells have been stimulated by an immunogenic composition, cells from asubject, or host, to which the immunogenic composition has beenpreviously administered, may be isolated. Genomic DNA or RNA may beextracted and sequenced using methods known in the art. Specificregions, such as the CDR may be analyzed to determine if certainsequences are present reflecting a response to an antigen. Furtherclonotype switching or affinity maturation may also be observed bysequencing and thereby also be indicative of an immune response.

Numerous methods of sequence determination are compatible with thesystems and methods of the disclosures. Exemplary methods for sequencedetermination include, but are not limited to, hybridization-basedmethods, such as disclosed in Drmanac, U.S. Pat. Nos. 6,864,052;6,309,824; and 6,401,267; and Drmanac et al, U.S. patent publication2005/0191656, which are incorporated by reference, sequencing bysynthesis methods, e.g., Nyren et al, U.S. Pat. Nos. 7,648,824,7,459,311 and 6,210,891; Balasubramanian, U.S. Pat. Nos. 7,232,656 and6,833,246; Quake, U.S. Pat. No. 6,911,345; Li et al, Proc. Natl. Acad.Sci., 100: 414-419 (2003); pyrophosphate sequencing as described inRonaghi et al., U.S. Pat. Nos. 7,648,824, 7,459,311, 6,828,100, and6,210,891; and ligation-based sequencing determination methods, e.g.,Drmanac et al., U.S. Pat. Appl. No. 20100105052, and Church et al, U.S.Pat. Appln Nos. 20070207482 and 20090018024.

Sequence information may be determined using methods that determine many(typically thousands to billions) nucleic acid sequences in anintrinsically parallel manner, where many sequences are read outpreferably in parallel using a high throughput serial process. Suchmethods include but are not limited to pyrosequencing (for example, ascommercialized by 454 Life Sciences, Inc., Branford, Conn.); sequencingby ligation (for example, as commercialized in the SOLiD™ technology,Life Technology, Inc., Carlsbad, Calif.); sequencing by synthesis usingmodified nucleotides (such as commercialized in TruSeq™ and HiSeg™technology by Illumina, Inc., San Diego, Calif., HeliScope™ by HelicosBiosciences Corporation, Cambridge, Mass., and PacBio RS by PacificBiosciences of California, Inc., Menlo Park, Calif.), sequencing by iondetection technologies (Ion Torrent, Inc., South San Francisco, Calif.);sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View,Calif.); nanopore-based sequencing technologies (for example, asdeveloped by Oxford Nanopore Technologies, LTD, Oxford, UK), and likehighly parallelized sequencing methods.

B. Therapeutic Applications

In other applications, the composition and methods of the disclosure maybe used for the generation of an immune response or desired antibody toa target epitope. Antibodies may be widely useful tools for a variety ofapplications in addition to vaccines.

In some instances, for example, antibodies may be generated as atherapeutic drug. Numerous antibody based therapies comprise specificmonoclonal antibodies directed towards a target molecule such as a cellreceptor. In this case, a target molecule, or target protein associatedwith a disease, may be selected as an antigen. In some cases, anantibody, using methods described herein, may be raised against thespecific target molecule. Target epitopes may be selected such that thebinding of an antibody may inhibit the function or enhance the functionof the target molecule. For example, antibody based therapies have beensuccessfully employed against cell receptors such as VEFG for a numerousindications. Binding of monoclonal antibodies to the VEGF receptor,inhibits the binding of VEGF ligand for VEGF receptor and for someindications producing a therapeutic effect. Using composition andmethods of this disclosure, a similar antibody, directed towards anyselected antigen or target molecule, may be developed for a therapeuticpurpose.

In other examples, an antibody therapeutic may be generated using thecompositions and methods of the disclosure. For example, an antibodygenerated towards an antigen associated with a specific pathology may bechosen. In one example, the membrane receptor HER2 may contain certainmutations that are associated with breast cancer. HER2 mutants (genevariants containing oncogenic mutations) may be selected as an targetantigen. In particular, a target epitope that is specific to the mutantHER2 variant may be chosen. Using compositions and methods of thisdisclosure, an antibody may be generated to a specific target epitope onthe mutant HER2 protein. In some examples, the antibody may be maderecombinantly, or generated in a mouse and further humanized foradministration in a human subject who may have or suspected of havingcancer. In some cases, an antibody therapeutic may be formulated wherebythe antibody gerenated by the compositions and methods of thisdisclosure may be associated with (e.g. covalently linked, orconjugated) an chemotherapeutic agent, such as taxol. In some cases, theantibody may be used to target the chemotherapeutic agent to cellsexpressing the mutant HER2 protein, thereby selectively killing thecancer cells. In other examples, the antibody generated against mutantHER2 may be used to block or inhibit stimulatory signals from binding tothe HER2 receptor and involved in maintaining the cancer cell.Inhibition of signaling through the HER2 receptor may decreasesurvivability or proliferation of the cancer cell.

In another example, the compositions and methods of this disclosure maybe used to formulate a therapeutic that may selectively bind, and insome example attenuate or kill, certain clonotypes of T cell or B cells.In one example, an immunogenic composition of the disclosure may beformulated to contact a specific clonotype of cells, which may useful inthe treatment of numerous diseases, such as autoimmune diseases,neurological diseases and inflammatory diseases. For example, in thecase of an autoimmune disease such as lupus, certain T cells or B cellsmay be secreting antibodies or inflammatory agents that cause thedisease. In some instances, certain T cell and B cell clonotypes maycontain receptors specific for certain antigens. In the case of lupus,these antigens may be “self antigens” or proteins found on the surfacesof healthy cells in a patient suffering from lupus. In some cases it maybe useful as a treatment to eliminate certain harmful clonotypes thatproduce antibodies and inflammatory agents in response to self antigens.

Using compositions and methods of the disclosure, an immunogeniccomposition may be formulated comprising self antigens recognized byspecific harmful B and/or T cell clonotypes. In some cases, theimmunogenic composition may comprise antigens associated with a toxin orchemotherapeutic for killing or attenuating B and T cells. Whenadministered to a subject, the immunogenic composition may bind to Band/or T cells. In some cases the associated chemotherapeutic or toxinmay selectively kill harmful immune cell clonotypes.

In another example, a therapeutic may also comprise an immune cell whichhas been trained to recognize a specific harmful antigen, such as thatfound in an infection, or from cancer. For example, immune cells may betrained to recognize and elicit a response to a cancer antigen such asProstate-specific antigen, a cancer antigen found in subsets of subjectswith prostate cancer. In some cases, immune cells may be harvested froma patient. An immunogenic composition of the disclosure, comprisingProstate-specific antigen variants may be administered to the immunecells. In some cases, APC cells, such as dendritic cells, may uptakemembers of the immunogenic composition and present peptides of theantigens on the surface. This may be achieved with techniques known inthe art, such as lipid mediated delivery, electroporation, recombinantviral delivery and the like. In some cases, B cells may be selected thatbind and secrete antibodies to the immunogenic composition. B cells maybe allowed to undergo SMD to generate antibodies with increasing bindingaffinity and/or specificity. In some cases, these immune cells, such asthe APCs, T cells or B cells may be administered back to a patient. Insome cases, such as in the example of APCs, T cell in the patient's bodywill elicit an immune response to cells expressing the Prostate-specificantigen, thus killing cancer cells.

In another example, a therapeutic may also comprise a recombinant virusencoding one or more antigen variants of an immunogenic composition. Insome cases, expression of antigen variant proteins may not be desirablebefore administration to a subject. Rather, nucleic acids encoding theantigen variants may be administered to a subject, whereby the antigenvariants proteins are generated by the subject or host.

C. Diagnostic and Research Tools

Further, generation of specific antibodies specific for a target epitopemay be useful as tools in diagnostics. Numerous cellular and biochemicalassays rely upon antibodies for sensitive detection of trace amount ofproteins. Various diagnostic tests ranging from HIV tests to tests forHashimoto's disease require diagnostic tests that comprise antibodies.The composition and methods of the disclosure provide for the generationof a desired antibody to a target epitope on any selected antigen. Theantibody may be used as a tool for any diagnostic test, biosensor,cellular or biochemical assay used in the detection of the antigen usedto raise the antibody.

For example, the immunogenic composition of the disclosure may be usedas as binding partner for certain antibodies. In one example, animmunogenic composition may be used to test the presence or absence ofcertain antibodies in the blood of a person having or suspected ofhaving HIV. The immunogenic composition may be used as bait to determinethe specificity and affinity of certain anti-HIV antibodies that may bepresent. In some examples, HIV antibodies present in blood may beisolated and complexed, in vitro, with one or more immunogeniccompositions comprising HIV antigens. Immunoassays such as ELISA orsurface Plasmon Resonance may be used to characterize the nature of theblood derived HIV antibodies. Information regarding the binding of theantigen variants of the immunogenic composition and the HIV antibodiesmay provide information about the amount of HIV antibodies present, thenature the antibodies (mature, vs. intermediate antibodies), theepitopes to which the antibodies have been generated the like. Thisinformation may be useful in the diagnosis and/or prognosis of HIVinfection or any other pathologies associated with the infection.

Similarly, antibodies are essential tools for basic research and may beused for applications related to experimentation. Antibodies generatedby the compositions and methods of the disclosure may be used forprotein purification, or characterization or quantification of proteinsof interest in basic research. The composition and methods of thisdisclosure provide for methods to generate a desired antibody for use astools in a variety of basic research tool applications, includingimmunoassays.

V. System for Data Transmittal and Storage

Methods and composition of this disclosure has the capability ofgenerating millions of bits of information pertaining to the design andformulation of various immunogenic compositions. The massive amount ofraw and processed data generated from the compositions and methods ofthe disclosure may be stored in any manner that allows for archiving andretrieval, most often through memory storage devices accessed bycomputer. Given that the compositions and methods of the disclosure maybe applied the development of vaccines, therapeutics diagnostics andother tools, there may be wide range of rules and regulations that maygovern the use and storage of these data. For clinical testing of humansubjects for example, appropriate consents must be obtained from partiesinvolved and standard HIPAA regulations will govern how this informationis stored and disseminated. In general, this information must beprotected from access by any unauthorized individual and may becommunicated from the clinical laboratory that performed the test onlyto the ordering physician or his/her designee in accordance with stateand federal laws. In most cases, the ordering physician then shares thisinformation with patients and medical staff who are directly involved inthe case. For analyses of nonhuman species and research applications, avariety of federal and state laws and regulations, policies of fundingagencies and institutional rules and regulations may impact how thisinformation is stored and disseminated.

Any appropriate method can be used to communicate information pertainingto data generated by the compositions and methods of the disclosure toanother person. For example, information can be given directly orindirectly to a professional, and a laboratory staff member can inputthe report of vaccine design into a computer-based record. In somecases, information is communicated by making a physical alteration tomedical or research records. For example, a medical professional maymake a permanent notation or flag a medical record for communicating therisk assessment to other medical professionals reviewing the record. Inaddition, any type of appropriate communication can be used tocommunicate the risk assessment information. For example, mail, e-mail,telephone, and face-to-face interactions can be used. The informationalso can be communicated to a professional by making that informationelectronically available to the professional. For example, theinformation can be communicated to a professional by placing theinformation on a computer database such that the professional can accessthe information. In addition, the information can be communicated to ahospital, clinic, or research facility serving as an agent for theprofessional. An exemplary diagram of computer based communication isshown in FIG. 4.

Another aspect of the invention provides a system that is configured toimplement the methods of the disclosure. The system can include acomputer server (“server”) that is programmed to implement the methodsdescribed herein. FIG. 4 depicts a system adapted to enable a user tostore, analyze, and process sequence information. The system includes acentral computer server that is programmed to implement exemplarymethods described herein. The server includes a central processing unit(CPU, also “processor”) which can be a single core processor, a multicore processor, or plurality of processors for parallel processing. Theserver also includes memory (e.g. random access memory, read-onlymemory, flash memory); electronic storage unit (e.g. hard disk);communications interface (e.g. network adaptor) for communicating withone or more other systems; and peripheral devices which may includecache, other memory, data storage, and/or electronic display adaptors.The memory, storage unit, interface, and peripheral devices can be incommunication with the processor through a communications bus (solidlines), such as a motherboard. The storage unit can be a data storageunit for storing data. The server is operatively coupled to a computernetwork (“network”) with the aid of the communications interface. Thenetwork can be the Internet, an intranet and/or an extranet, an intranetand/or extranet that is in communication with the Internet, atelecommunication or data network. The network in some cases, with theaid of the server, can implement a peer-to-peer network, which mayenable devices coupled to the server to behave as a client or a server.In some embodiments, the computing resources can be configured into acloud-service model.

The storage unit can store files, such as sequence data, sample data,molecular barcodes, software, or any aspect of data associated with theinvention. The data storage unit may be coupled with data that can binsample sequence with the sample source or other information contained ina molecular barcode.

The server can communicate with one or more remote computer systemsthrough the network. The one or more remote computer systems may be, forexample, personal computers, laptops, tablets, telephones, smart phones,or personal digital assistants. The remote computer systems may, forexample, be used to transmit patient data to a caregiver. The data orhardware or system, for example, may be encrypted or modified (e.g. tocomply with HIPPA rules and standards).

In some situations the system includes a single server. In othersituations, the system includes multiple servers in communication withone another through an intranet, extranet and/or the Internet.

The server can be adapted to store sample information, such as, forexample, sample source, date, orientation, sequence, statistical data,or any other information of potential relevance. Such information can bestored on the storage unit or the server and such data can betransmitted through a network.

Methods as described herein can be implemented by way of machine (orcomputer processor) executable code (or software) stored on anelectronic storage location of the server, such as, for example, on thememory, or electronic storage unit. During use, the code can be executedby the processor. In some cases, the code can be retrieved from thestorage unit and stored on the memory for ready access by the processor.In some situations, the electronic storage unit can be precluded, andmachine-executable instructions are stored on memory. Alternatively, thecode can be executed on a second computer system.

Aspects of the systems and methods provided herein, such as the server,can be embodied in programming. Various aspects of the technology may bethought of as “products” or “articles of manufacture” typically in theform of machine (or processor) executable code and/or associated datathat is carried on or embodied in a type of machine readable medium.Machine-executable code can be stored on an electronic storage unit,such memory (e.g., read-only memory, random-access memory, flash memory)or a hard disk. “Storage” type media can include any or all of thetangible memory of the computers, processors or the like, or associatedmodules thereof, such as various semiconductor memories, tape drives,disk drives and the like, which may provide non-transitory storage atany time for the software programming. All or portions of the softwaremay at times be communicated through the Internet or various othertelecommunication networks. Such communications, for example, may enableloading of the software from one computer or processor into another, forexample, from a management server or host computer into the computerplatform of an application server. Thus, another type of media that maybear the software elements includes optical, electrical, andelectromagnetic waves, such as used across physical interfaces betweenlocal devices, through wired and optical landline networks and overvarious air-links. The physical elements that carry such waves, such aswired or wireless likes, optical links, or the like, also may beconsidered as media bearing the software. As used herein, unlessrestricted to non-transitory, tangible “storage” media, terms such ascomputer or machine “readable medium” refer to any medium thatparticipates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, tangible storage medium,a carrier wave medium, or physical transmission medium. Non-volatilestorage media can include, for example, optical or magnetic disks, suchas any of the storage devices in any computer(s) or the like, such maybe used to implement the system. Tangible transmission media caninclude: coaxial cables, copper wires, and fiber optics (including thewires that comprise a bus within a computer system). Carrier-wavetransmission media may take the form of electric or electromagneticsignals, or acoustic or light waves such as those generated during radiofrequency (RF) and infrared (IR) data communications. Common forms ofcomputer-readable media therefore include, for example: a floppy disk, aflexible disk, hard disk, magnetic tape, any other magnetic medium, aCD-ROM, DVD, DVD-ROM, any other optical medium, punch cards, paper tame,any other physical storage medium with patterns of holes, a RAM, a ROM,a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, acarrier wave transporting data or instructions, cables, or linkstransporting such carrier wave, or any other medium from which acomputer may read programming code and/or data. Many of these forms ofcomputer readable media may be involved in carrying one or moresequences of one or more instructions to a processor for execution.

The results of sequencing can be presented to a user with the aid of auser interface, such as a graphical user interface.

VI. Examples

It will be understood by those of skill in the art that numerous andvarious modifications can be made to yield essentially similar resultswithout departing from the spirit of the present disclosure. All of thereferences referred to herein are incorporated by reference in theirentirety for the subject matter discussed. The following examples areincluded for illustrative purposes only and are not intended to limitthe scope of the disclosure.

Example 1: Method of Obtain Structural Models of Variable SurfaceGlycoprotein

To get a structural model of a Variable Surface Glycoprotein, 37structural representatives from NCBI NR database are aligned. From thealignment a profile Hidden Markov Model is generated using the programHMMer3. The HMM is used to detect homologous structures in PDB with aminimum e-value of 1×10⁻³. The HMM is used to align the structures'sequence to the reference sequence. An expected minimum threshold of 30%identity between at least one member of the antigen variants is set toassert sufficient homology. No more than 10% insertions/deletions in thelocal alignment region are permitted.

Example 2: Method for Diversifying TCR Epitopes

In this example, a library of antigenic variants is prepared, optimizedfor diversifying TCR epitopes. For diversifying a set of homologs in thenon target epitope regions, antigen homologs are identified from areference database for the antigen HIV gp120 protein. The antigens arealigned using the program MUSCLE. An HMM is generated using alignmentdata and the program HMMer. Structural models are obtained by searchingPDB with the HMM generated from the alignment data. Structural model arealigned to homologs using HMM and a PWM is extracted from the alignment.Each position in the structures are mapped to a position in thealignment, a column in the HMM, and a column in the PWM. Map surfaceaccessibility is determined for each column in the PWM from thestructure by Modeller. MHC-II TCR epitopes are identified fromliterature and mapped to the PWM. Surface exposed residues arediversified. MHC-II TCR epitopes are optimized. Non-exposed TCR epitopesare masked so they are not further diversified. Optimizing dispersion isperformed on the surface exposed residues by manipulating diversityfrequencies in the remainder of the positions that are surface exposedand not part of TCR epitopes. At any time, the PWM is now a design thatcould be synthesized to produce an antigen library. A collection of10000 sequences is sampled from the design in-silico by bioinformaticssimulation and analyzed. If the sequences are either so similar to oneanother that they contain many off-target epitopes in common, or sodistant from the reference structure that they have a low probability offolding, the PWM is altered and retested. Optimization is performed bylinear scaling of non-dominant amino acid frequencies compared todominant amino acid frequencies at each position. During testing, 90% ofa set of 100 simulated molecules are observed to be greater than 40%identical to a known reference sequence, and no off-target epitope,defined as any collection of surface exposed residues within a 25 squareangstrom radius centered on the carbon alpha backbone of a residue alsoon the surface, has a percent identity greater than 90% to any othermember by pairwise comparison. If these criteria aren't met, the PWM isaltered to be either more or less diverse and process repeated. As analternative to linear scaling, a random Monte Carlo stochastic sampling,a genetic algorithm, or manual intervention could also be used tooptimize the final PWM.

Example 3: Method for Optimizing BCR and TCR Epitopes in HIV Gp120Protein

In this example, a library of antigenic variants is prepared, optimizedfor diversifying TCR epitopes. For diversifying a set of homologs in thenon target epitope regions: first identify antigen homologs from areference database for the antigen HIV gp120 protein. The antigens arealigned using the program MUSCLE. An HMM is generated using alignmentdata and the program HMMer. Structural models are obtained by searchingPDB with the HMM generated from the alignment data. Structural model arealigned to homologs using HMM and a PWM is extracted from the alignment.Each position in the structures are mapped to a position in thealignment, a column in the HMM, and a column in the PWM. Map surfaceaccessibility is determined for each column in the PWM from thestructure by Modeller. MHC-II TCR epitopes from literature are mapped tothe PWM. Non-exposed residues are assigned as reference sequence. Tooptimize MHC-II TCR epitopes, a computational mask is placed onnon-exposed and TCR epitopes so they are not further diversified.Optimized dispersion is performed by manipulating diversity frequenciesin the remainder of the positions that are surface exposed and not partof TCR epitopes. At any time, the PWM is now a design that could besynthesized to produce an antigen library. A collection of 1000000sequences can be sampled from the design in-silico by bioinformaticssimulation and analyzed. If the sequences are either so similar to oneanother that they contain many off-target epitopes in common, or sodistant from the reference structure that they have a low probability offolding, the PWM is altered and retested. Optimization is performed bylinear scaling of non-dominant amino acid frequencies compared todominant amino acid frequencies at each position. During testing, 80% ofa set of 1000 simulated molecules are greater than 25% identical to aknown reference sequence, and no off-target epitope, defined as anycollection of surface exposed residues within a 100 square angstromradius centered on the carbon alpha backbone of a residue also on thesurface, and have a percent identity greater than 90% to any othermember by pairwise comparison. If these criteria aren't met, the PWM isaltered to be either more or less diverse. As an alternative to linearscaling, a random Monte Carlo stochastic sampling, a genetic algorithm,or manual intervention could also be used to optimize the final PWM.

Example 4: Method for Vaccine Development

In the case of generating one or more ensembles for the generation of avaccine, specific algorithms and methods may be used. For example, asequence database of all known variants for a target antigen, such as HAprotein is aligned and rendered non-redundant at 90% identity. Apositional weight matrix (PWM) is extracted from the alignment, toobtain a polymorphism compendium of all tolerated amino acids at allpositions and their frequencies. A library design is optimized, using asinput the PWM, applying amino acid variation to a reference H1N1 or H3N2virus or B scaffold and eliminating length variation inconsistent withthe reference scaffold. The scaffold is then diversified, preferentiallyweighting diversity to residues determined to be at least partiallysolvent accessible. To optimize the library, diversity in solventaccessible positions is then linearly scaled, with proportions of themost dominant “WT” residue compared to alternative diversity modified inorder to optimize a library dispersion target according to the followingscoring function: return true if <65% identity of any 25 Angstromdiameter surface “candidate epitope” when comparing any two variantsfrom a randomly sampled set of 1000 selected members; else false. Aparallel optimization cycle ensures maximum display of immunodominantMHC-I and MHC-II epitopes in the resulting molecules, preferably inconserved core regions shared among members. Scoring function isΣ((probability of peptide display by MHC display prediction)*(frequencyof MHC carriers in population)*(probability of peptide in ensemble)).

The resulting library is then be expressed on Mammalian display (orPhage, Hyperphage, Yeast) and selected against broadly neutralizingantibodies (bnAbs) against known broadly neutralizing epitopes to selectfor variants still able to adopt native fold and present the broadlyneutralizing epitopes. Residual diversity is evaluated byhigh-throughput sequence analysis of post-selection positive populationscompared to library.

Provided sufficient residual diversity exists, additional rounds arefurther selected in the presence of heat, intercalating agents and otherprotein folding selective pressures. From the final selected pool ofbnAb+/stability+ members, a set of 100 antigen variants exhibitingmaximal epitope dispersion (i.e. most different from one another) willbe selected, based on 10,000 Monte Carlo random starts of greedyaddition of greatest distance members from the source pool into thefinal selection set, using the aforementioned function for optimization(i.e. pick a random member, then keep adding new members that are as faraway from all of the previously selected members in the set as possible,and as far away from seasonal wt strains as possible, until 100 membersis reached. This is performed 10,000 times, and then checked to seewhich set is the most dispersed).

The resulting 100 members are then expressed and tested to confirmfolding and bnAb binding in the terminal expression system anticipatedfor production. A population of antigens is purified without His orother tags using bnAb affinity columns/beads/etc. The resulting purepools are then combined into single ventivalent (20×), quinquavalent(50×), and centivalent (100×) pools composed of equal composition ofeach antigen. The performance of the vaccine is validated using FACS andan animal model.

FACS: A population of cells containing known bnAbs (a library ofantibodies with spike in of broadly neutralizing B-cells, or apopulation of B-cells from an individual known to carry broadlyneutralizing B-cells) is sorted, using the ensemble as a stainingreagent. A single seasonal strain can act as a second color control todistinguish strain-specific B-cells from broadly elicited B-cells.Xivalent-H+++ cells are characterized individually for epitope by SPR inbinning studies.

Animal model validation: Animal models (mice, ferrets, primates, orhumans) are provided either a standard tri-valent, an Xivalent, or asaline/adjuvant control. Xivalent doses are made such that theindividual components are below the minimum concentration required foran effective immune response. Animals are subjected to cross-viralchallenge against a novel strain not found in any of the vaccines, aswell as direct viral challenge to the “seasonal” found in the trivalentstandard of care. Responses are assayed by ELISA, FACS, viral titers,animal disease/fatality score, and high throughput sequencing of the BCRand TCR responding populations. In some cases, an antiviral agent mayalso be administered in addition to the tri-valent or Xivalent vaccine.

Example 5: Generation of HA Protein Ensembles

6500 hemagglutinin variants are obtained from the public record.Variants are all aligned to a profile using a Hidden Markov Model,rendered non-redundant at 95% identity, and analyzed for positionalconservation using the Simpson's index. The resulting conservationprofile is then displayed on a reference structure of hemagglutinin asshown in FIG. 3. The black/dark grey regions are regions that varyeasily across different strains (300). Most regions of the proteinappear non conserved as reflected in black/dark grey. Certain areas,marked in lighter grey (301), are conserved regions or regions that maybe referred to as “broadly neutralizing stem” regions. This analysisnaturally identified these regions based on sequence data. Formally, itidentified a single patch (3×, given a trimer) of 50 Å², with highconservation profiles statistically discriminated above backgroundperfectly overlaps a known broadly neutralizing epitope. This analysisis also a direct reflection of an epitope suitable for drug targeting ora an epitope as detected by a host immune system: the grey hotspots athigh concentrations, and the dark regions at low concentrations.

Specifically, to generate this map of HA protein conservation, a largedataset of diverse sequences of the target antigen, or 6500 sequences ofhemagglutinin are obtained. Next, a representative with a solved crystalstructure is identified. The sequences are then aligned with thesequence of the crystal structure.

A profile hidden Markov Model to represent diverse HA protein is used toalign the sequences accurately via the Viterbi algorithm. The resultingdataset has redundant sequences removed to reduce bias. For example, theHA dataset is reduced to 95% identity. A positional weight matrix (PWM)is obtained from the alignment that summarizes the frequency of allamino acids at all positions in the alignment. The PWM is filtered toremove positions that are mostly gap states (unusual inserts), andfiltered to remove minority gap states by renormalizing the amino acidvectors in match-state dominant position. For example, mostly gappypositions in the HA alignment are removed, and the PWM are adjusted sothat no positions show a chance of having “no amino acid” as an option.Every sequence that the PWM represented is made the same length. Thislength, and the consensus, is proximal to a variant known to fold. ThePWM is then considered a library design. It contains instructions forwhat amino acids to appear at what positions at what frequencies.

Different antigen variants are then optimized with diversity in thedesign. A target dispersion of molecules in a given library size aretaken as input, and the individual diversity vectors are uniformlyscaled during iterations to produce a resulting library that emits asequence distribution at the desired dispersion state. For example alibrary of 100 million HA variants on yeast display are panned. All ofthe molecules are selected to differ by at least 10% from the nextclosest member, and have an average distance of 50% identity across thelibrary. The algorithm is then “stretched” the amino acid diversity atall positions until that criteria was met.

Additional selection steps included rational modification of criticalsites to improve library performance. For example, some mutationcombinations are not predicted to work in nature, so they are removedthem from the library. The target epitope is also optimized to maintainmore conservation on purpose. Similiarly the core of the protein is alsooptimized to maintain more conservation, and leave diversitypredominantly in surface exposed residues. Additionally, rationallyencoded CD4+ and CD8+ specific T-cell epitopes known to aid inelicitation into the core of the protein are also added to the design.

The library is then commercially synthesized and produced. The PWM issent to a synthesis company to produce a library bearing the diversityof the design. The library may be sent to TRIM synthesis (Mohan,Glanville etc 2013) or Isogenica 6tuple synthesis (Zhai & Glanville2011).

The library is then displayed on phage. We attach fluorophores to manyantibody variants known to recognize the broadly neutralizing epitope,and then sort the library using this color marker. Only HA variants thatcan still be recognized by broadly neutralizing antibodies of manysources are selected out of the library

Temperature selection of the library. The experiment from the previousstep is repeated at higher temperatures and in the presence ofdestabilizing agents in order to select for very stable versions of HAthat will be easier to work with during manufacturing.

An immunogenic composition for a vaccine is formulated for flu based onthe designed ensemble.

Example 6: Generation of a KRAS-Mutant Vaccine Therapeutic

The KRAS receptor is a signaling receptor often mutated in variouscancers, including lung cancer and colorectal cancers. 100 KRAS variantsincluding the commonly oncogenic KRAS P.G12D variant, are obtained fromthe public record. Variants are all aligned to a profile using a HiddenMarkov Model, rendered non-redundant at 95% identity, and analyzed forpositional conservation using the Simpson's index. The resultingconservation profile is then displayed on a reference structure of KRAS.Most regions of the protein appear conserved in certain areas. In someareas, non conserved substitutions are found, including the P.G12Dmutation. This analysis naturally identifies these regions based onsequence data.

Specifically, to generate the map of KRAS protein conservation, a largedataset of diverse sequences of the target antigen, or 100 sequences ofKRAS are obtained. Next, a representative with a solved crystalstructure was identified. The sequences were then aligned with thesequence of the crystal structure.

A profile hidden Markov Model to represent diverse KRAS protein is usedto align the sequences accurately via a neural network algorithm. Theresulting dataset had redundant sequences are removed to reduce bias.For example, the KRAS dataset is reduced to 95% identity. A positionalweight matrix (PWM) is obtained from the alignment that summarizes thefrequency of all amino acids at all positions in the alignment. The PWMis filtered to remove positions that are mostly gap states (unusualinserts), and filtered to remove minority gap states by renormalizingthe amino acid vectors in match-state dominant position. For example:mostly gappy positions in the KRAS alignment were removed, and the PWMis adjusted so that no positions show a chance of having “no amino acid”as an option. Every sequence that the PWM represents is made the samelength. This length, and the consensus, is proximal to a variant knownto fold. The PWM was then considered a library design. It containedinstructions for what amino acids to appear at what positions at whatfrequencies. An epitope containing the KRAS P.G12D mutation is selectedas the target epitope.

Different antigen variants are then optimized with diversity in thedesign. A target dispersion of molecules in a given library size aretaken as input, and the individual diversity vectors are uniformlyscaled during iterations to produce a resulting library that emits asequence distribution at the desired dispersion state. For example alibrary of 1 million KRAS variants on yeast display was panned. All ofthe molecules were selected to differ by at least 10% from the nextclosest member, and have an average distance of 50% identity across thelibrary. The algorithm then “stretches” the amino acid diversity at allpositions until that criteria is met.

Additional selection steps include rational modification of criticalsites to improve library performance. For example, some mutationcombinations are not predicted to work in nature, so they are removedthem from the library. The target epitope is also optimized to maintainmore conservation on purpose. Similiarly the core of the protein wasalso optimized to maintain more conservation, and leave diversitypredominantly in surface exposed residues. Additionally, rationallyencoded CD4+ and CD8+ specific T-cell epitopes known to aid inelicitation into the core of the protein are also added to the design.

The library is then commercially synthesized and produced. The PWM issent to a synthesis company to produce a library bearing the diversityof the design. The library may be sent to TRIM synthesis (Mohan,Glanville etc 2013) or Isogenica 6tuple synthesis (Zhai & Glanville2011).

The library is then displayed on phage are various temperatures andrepeated at in the presence of destabilizing agents in order to selectfor very stable versions of KRAS that will be easier to work with duringmanufacturing.

Each KRAS variant is scored based on biochemical stability and theability to be expressed. After scoring, an immunogenic composition isformulated based on the designed ensemble. Various KRAS variants areselected from the library based on biochemical stability. The moststable molecules are generated and combined in various ratios, such thateach antigen in the immunogenic composition is not, in isolation, at aconcentration capable of eliciting an immune response when administeredin a subject.

An immunogenic composition is formulated using various KRAS antigenvariants. Various targeting molecules are also included, such as apeptide that binds CD138 (Syndecan-1, Heparan sulfate proteoglycan) forspecific targeting of the immunogenic to B cells, as well as an apatmerthat binds CD25 (Tac antigen, IL-2Rα, p55), for for specific targetingof the immunogenic composition to T cells.

The immunogenic composition is then administered subcutaneously, withacceptable pharmaceutical carriers, into a human patient having orsuspected of having a cancer resulting from KRAS P.G12D mutation.

The patient's immune cells produce both an antibody and cytotoxic T cellresponse to cancer cells containing the KRAS P.G12D mutation. B-cell andT-cells of the patient do not ellict an immune response to non-targetepitopes, such as wildtype KRAS receptors (e.g. receptors not containingthe KRAS P.G12D mutation.

Example 7: Generation of a KRAS P.G12D Antibody Therapeutic

In an alternative example, the immunogenic composition formulated usingvarious KRAS P.G12D antigen variants of Example 6 may be used togenerate a therapeutic antibody. In this case, the immunogeniccomposition may be administered to a mouse or rabbit hybridoma cells.Hybridoma cells that produce an antibody that is specific for the KRASP.G12D epitope are selected. Further rounds of antibody optimization mayalso be included, whereby hypersomatic mutation allows for improvedbinding affinity of the antibody to the target epitope.

Nucleic acids from hybridomas producing selective antibodies to the KRASP.G12D may be sequenced to determine the coding sequence for theantibodies. Nucleic acids may be cloned into expression vectors and thesequence further optimized. The sequence may be mutated to incorporatemutations to “humanize” the antibody as known in the art.

Humanized recombinant antibodies may be recombinantly expressed andpurified from in vitro cell culture lines, such as HEK293 cells. Atherapeutic antibody formulation is generated by conjugating the KRASP.G12D antibody to a chemotherapeutic agent, such as cisplatin. Thetherapeutic agent may be then administered subcutaneously, withacceptable pharmaceutical carriers, into a human patient having orsuspected of having a cancer resulting from KRAS P.G12D mutation. Theantibody formulation specifically binds to cancer cells expressing theKRAS P.G12D receptor. The attached chemotherapeutic selectively killsthose cells.

Example 8: Generation of a KRAS P.G12D Immune Cell Therapeutic

In an alternative example, the immunogenic composition formulated usingvarious KRAS P.G12D antigen variants of Example 6 may be used togenerate an immune cell therapeutic. In this case, the immunogeniccomposition may be administered to one or more immune cells harvestedfrom a patient in need of treatment or prevention of cancer containingthe KRAS P.G12D mutation. Dendritic cells (DCs) may be harvested from apatient and cultured in vitro. The immunogenic composition of Example 6is administered to the DCs. In some examples, DCs are electroporated toallow for antigens of the immunogenic composition to be taken up andpresent on MHC molecules. In other examples, nucleic acids encodingantigen variants are administered to the DCs, such as through lipidbased transfer or through recombinant viral delivery vectors. Additionalagents may be used to help ellict MHC presention of the KRAS P.G12Dantigen variants.

DCs cells are tested using a range of immune assays including ELISA orFACs to observe presentation of the KRAS P.G12D antigens. DCsdemonstrating this presention may then be administered to the subjectfrom whom the cells were originally derived, or administered to adifferent subject in need of treatment.

Once inside the subject, DCs may allow for the subject to elict animmune response to KRAS P.G12D antigens, either through T cell or B cellresponse pathways.

Example 8: Generation of a KRAS P.G12D DNA Vaccine

In an alternative example, the immunogenic composition formulated usingnucleic acids encoding various KRAS P.G12D antigen variants of Example 6may be used to generate a therapeutic. In this case, the immunogeniccomposition comprises a plurality of nucleic acids may encoding one ormore proteins sequence of KRAS antigenic variants. In one examplenucleic acids are conjugated to a suitable vector, such as lipid basedelivery vehicle to be administered into patient in need of treatment orprevention of cancer containing the KRAS P.G12D mutation. In anotherexample, the nucleic acids encoding the immunogenic composition may bedelivered via recombinant viral vectors.

Once inside the subject, nucleic acids encoding the immunogeniccomposition may enter the subject's or host cells. In some cases, thecells are immune cells. In some cases, the nucleic acids are targeted todiseased cells. In the case of diseased cell, the cells uptake thenucleic acids. Inside the host cells, the nucleic acids are transcribedand/or translated to produce antigen variant proteins. In one example,the antigen proteins may be secreted by the cells, or displayed on theoutside of the cell. The host's or subject's immune system generates animmune response to cells expressing the antigen variants of theimmunogenic composition, thus killing the diseased or cancer cells. Theimmune response may be generated through innate, humoral or adaptiveresponses.

Example 9: Select for Epitope-Specific Repertoires in a Phage Display,Yeast Display, CIS Display

A target epitope is known for an antigen of interest, and it isdesirable to engineer antibodies against that epitope, while minimizingtime spent characterizing antibodies against other epitopes. A set of 6or more antigen variants are designed that share the conserved epitopebut differ in other epitopes. The antigen pool is produced and presentedin parallel during selection, either against an unselected library, or alibrary already exposed to one or more enrichment rounds against thenative antigen. The total antigen concentration is limited duringenrichment to simultaneously select for epitope specificity andoff-rate. Non-labeled competitor wildtype can be introduced duringincubation steps to further select for epitope specificity and off-rate.Some of the antigens can be presented in parallel during selection foroptimization purposes, such as through phage display, yeast display, CISdisplay, or mammalian display. High throughput sequencing of enrichmentround pools can aid in discovery or detection of specific selectivity ofclonotypes.

Example 10: Select Epitope-Specific Repertores by Flow Cytometry ofHuman Cells or Mass Cytometry of Human Cells

A target epitope is known for an antigen of interest, and it isdesirable to recover antibodies against the epitope. A set of 6 or moreantigen variants are designed that share the conserved epitope butdiffer in other epitopes. Each antigen could optionally be provided adifferent marker, up to the limiting number of available markers.Antigens can also be pooled on the same markers. The cells are incubatedwith the labeled antigens and then sorted by cytometry. The number ofmarkers on a cell, as well as the intensity of decoration of that cell,can both be used to select for broadly reactive epitope binders. In someexamples, the markers represent fluorophores or combinations offluorescent signals that be detectable using flow cytometry or masscytometry of cells.

Example 11: Development of Bi-Specific T-Cell Engagers

The methods and compositions of the disclosure are used to develop aBi-specific T-cell engagers (BiTEs) a a class of artificial bispecificmonoclonal antibodies that are investigated for the use as anti-cancerdrugs. They direct a host's immune system, more specifically the Tcells' cytotoxic activity, against cancer cells.

BiTEs are fusion proteins consisting of two single-chain variablefragments (scFvs) of different antibodies, or amino acid sequences fromfour different genes, on a single peptide chain of about 55 kilodaltons.

The compositions and methods of the disclosure may be used to developantibody or antibody fragments for a BiTE molecule. For example, thecompositions and methods of the disclosure are used to generate anantibody toward CD3, a specific T cell marker as described herein. Theother antibody of the BiTE molecule may be an antibody generated towardsa tumor antigen (such as HER2). Thus one of the scFvs is designed tobinds to T cells via the CD3 receptor, and the other to a tumor cell viaa tumor specific molecule, such as HER2, bringing the T cell intoproximity with the cancer cell. The T cell is then activated to kill thecancer cell.

1-32. (canceled)
 33. A method for eliciting an immune response in a human subject, the method comprising: delivering at least six antigens to the human subject, wherein each of the at least six antigens comprises: a target epitope that is common to each of the at least six antigens; and one or more non-conserved regions that are outside of the target epitope; wherein the at least six antigens are delivered such that each individual antigen of the at least six antigens is delivered in an amount that is insufficient to be immunogenic to the human subject on its own, while the target epitope is delivered in an amount that is sufficient to generate an immune response in the human subject.
 34. The method of claim 33, wherein the at least six antigens are delivered as a single composition.
 35. The method of claim 33, wherein each of the at least six antigens comprises a peptide backbone.
 36. The method of claim 33, wherein, for each of the at least six antigens, the target epitope comprises a plurality of surface-exposed amino acids that are adjacent to one another in tertiary space, but discontinuous from one another in a primary sequence of the antigen.
 37. The method of claim 36, wherein the surface-exposed amino acids that are adjacent to one another in tertiary space have a surface-exposed surface area of at least 25 Å².
 38. The method of claim 36, wherein the surface-exposed amino acids that are adjacent to one another in tertiary space have a surface-exposed surface area of at most 2000 Å².
 39. The method of claim 36, wherein the surface-exposed amino acids that are adjacent to one another in tertiary space have a surface-exposed surface area of between 100 Å² and 1500 Å².
 40. The method of claim 33, wherein each of the at least six antigens share a common protein fold.
 41. The method of claim 33, wherein each of the at least six antigens comprises at least 100 amino acid residues.
 42. The method of claim 33, wherein each of the at least six antigens is from about 5 kDa to about 1000 kDa in size.
 43. The method of claim 33, wherein the common target epitope is at least 90% identical across each of the at least six antigens.
 44. The method of claim 33, wherein no antigen of the at least six antigens has more than 90% of its surface-exposed amino acids in common with any of the remaining at least six antigens.
 45. The method of claim 33, wherein the immune response comprises an interaction of a CD4+ T-cell with an antigen-class II WIC molecule complex presenting the common target epitope.
 46. The method of claim 33, wherein the subject has a disease selected from the group consisting of infectious disease, autoimmune disease, inflammatory disease, neurological disease, addiction, cardiovascular disease, endocrine disease and cancer.
 47. The method of claim 33, wherein a first antigen protein of any of the at least six antigens is immunologically cross reactive with an antibody raised against a second antigen protein of any of the at least six antigens.
 48. The method of claim 33, wherein the at least six antigens comprises at least 10 different antigens.
 49. The method of claim 33, wherein each of the at least six antigens have a binding equilibrium dissociation constant to an antibody produced by the subject that is less than 10⁻⁷M.
 50. The method of claim 33, wherein delivering at least six antigens to the human subject comprises delivering one or more nucleic acids that encode each of the at least six antigens.
 51. The method of claim 50, wherein delivering the at least six antigens to the human subject comprises further comprises delivery of the one or more nucleic acids via a viral victor or a lipid-containing complex.
 52. The method of claim 33, wherein delivering the at least six antigens comprises delivery of antigens via one or more or parenteral delivery, topical delivery, pulmonary delivery, intranasal delivery, vaginal delivery, enteral delivery, rectal delivery, oral delivery, or sublingual delivery. 